System and method for detecting multiple-excitation-induced light in a flow channel

ABSTRACT

A system for detecting signal components of light induced by multiple excitation sources, which includes a flow channel having two spatially separated optical interrogation zones; a light illumination subsystem that directs each of two light beams of different wavelengths to a different zone of the optical interrogation zones; a detector subsystem that converts detected light into a total electrical signal having both modulated and unmodulated electrical signals; and a processor that determines signal components from the light detected from each of the optical interrogation zones.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/657,845, filed Oct. 22, 2012, which claims benefit of priority toU.S. provisional patent application No. 61/550,243, filed Oct. 21, 2011;the contents of each are herein incorporated by reference in theirentirety.

TECHNICAL FIELD

This invention relates generally to the field of detecting light emittedfrom fluorescent molecules excited by multiple excitation light sources,and more specifically to a system and method that incorporates at leastone intensity modulated excitation light source and a nonmodulated lightsource to induce the emission of light from at least two spatiallyseparated optical interrogation zones of a flow channel and thedetection and measurement thereof.

BACKGROUND OF THE INVENTION

Flow cytometer is an instrument for measuring/analyzing individualfluorescently-labeled particles/cells being lined up and flown through aflow channel under hydrodynamic focusing or other focusing forces. Lightbeam from individual light sources (e.g. laser) of particularwavelengths is shaped typically to an elliptical or rectangular shapewith the major/long axis (about 50 to 200 microns) perpendicular to theflowing direction of the particles/cells in a flow channel andminor/short axis (about 10 to 30 microns) parallel to the flowingdirection of the particles/cells and is guided/directed to opticalinterrogation zones (OIZ) in the flow channel. As thefluorescently-labeled particles/cells pass through the light beamone-by-one in the optical interrogation zones, multiple physicalcharacteristics of single cells can be detected and measured. Theproperties measured include a particle's relative size (Forward Scatter,i.e. FSC), relative granularity or internal complexity (Side Scatter,i.e. SSC), and relative fluorescence intensity (i.e., fluorescencesignals from fluorescent molecules in the labeled cells under excitationby light sources.). These characteristics are determined using anoptical-to-electronic coupling system (i.e. photodetector) that recordshow the cell or particle scatters incident laser light and emitsfluorescence.

Traditionally, two approaches have been implemented for flow cytometerswith multiple-excitation light sources. In the first approach, theshaped elliptical laser beams from multiple sources (e.g., S1, S2 inFIG. 1) of different wavelengths propagate across the flow channel andform two optical interrogation zones (OIZ) at different verticallocations along the flow channel, spaced at certain distance in therange of, e.g., 100 to 200 microns. Thus when a cell flows through theflow channel, it will pass the individual OIZ (e.g. OIZ1 correspondingto laser beam S1 and OIZ2 corresponding to laser beam S2 in FIG. 1) insequence. Consequently, light in the laser beams will be scattered andfluorescent light will be emitted as well by different fluorescentmolecules (FM) possessed by the cell. Multiple fluorescent signals (FS)with different peak wavelengths may be emitted by different molecules atone OIZ. For example, S1 can be 488 nm laser and S2 can be 640 nm laser.Fluorescent molecules FITC, PE, and PE-Cy7 can be excited by S1 (488 nm)and emit light at peak wavelength of 519 nm, 578 nm, and 785 nmrespectively whilst APC and APC-Cy7 can be excited by S2 (640 nm) andemit light at peak wavelength of 660 nm and 785 nm respectively. It ispossible that the spectra of the fluorescent signals excited bydifferent light sources are overlapped (e.g. PE-Cy7 and APC-Cy7 are bothemitted at a peak wavelength of 785 nm, but need to be excited bydifferent wavelengths). Therefore, it would be essential that thefluorescent signals excited by different light sources (e.g. FS1 beingexcited by S1 and FS2 being excited by S2 in FIG. 1) are separated bythe light collection and separation optics for effective detection ofthese fluorescent signals. Otherwise, the flexibility of choosing thefluorochrome for cell staining would be limited and it becomes ashortcoming for a flow cytometer. Through light collection/separationoptics, the fluorescent signal FS1 and FS2 is collected and separated todifferent physical positions with a spatial distance large enough toaccommodate the filters and photodetectors for light splitting,filtering, and detection. This light splitting, filtering, and detectionsystem resolves the quantities of each corresponding fluorescentmolecule, thus are referred to fluorescence (FL) channels in flowcytometry. For example, fluorescent signal FS1 emitted from OIZ1 is thenfurther split and detected by photodetector D1 and D2 at two differentemission wavelengths to resolve the quantity of fluorescent molecule FM1(e.g., FITC) and FM2 (e.g., PE-Cy7) respectively and fluorescent signalFS2 emitted from OIZ2 is then further split and detected byphotodetector D3 and D4 at two different emission wavelengths to resolvethe quantity of fluorescent molecule FM3 (e.g., APC) and FM4 (e.g.,APC-Cy7). Photodetectors convert the detected fluorescent signals intoelectronic signals. Using electronic processors, such electronic signalsare then filtered, amplified and converted into digital signals, andfurther processed to derive various characteristics of eachcorresponding fluorescent molecule. For example, output E1 and E2correspond to the signals of FM1 and FM2 respectively and output E3 andE4 correspond to the signals of FM3 and FM4 respectively. When aparticle/cell passes through an OIZ, an electronic pulse will begenerated at the corresponding detection channel and such cell/particleinduced electronic pulse is characterized by its height, area, and widthto reveal the property of the particles/cells. For example, the heightof the pulse detected by one FL channel provides a good indication ofthe intensity of the corresponding fluorescent molecule. In summary, ina flow cytometer with a system configured shown in FIG. 1 and asdescribed above, fluorescent signals excited by different light sourcesis physically/optically separated for detection with differentphotodetectors, and fluorescent molecules with overlapped emissionspectra could be used to label the particles/cells in the sameexperiment.

However, the first approach has several limitations. (1) Complex opticssystem are needed to collect and to separate light from different OIZsin order to achieve an efficient and clear separation of light emittedfrom each OIZ with small physical separation distances of about 100-200microns. (2) Accurate control and delivery of excitation beams to theflow channel is required to have an accurate control of separationdistance between OIZs. In a flow cytometer, an individual cell isusually transported through the flow channel at a constant speed, whichmeans that the time interval between the detected digital pulsegenerated in OIZ1 and the detected digital pulse generated in OIZ2 willbe fixed. Therefore, in order to correlate signals from the same cellpassing through different OIZs, this time interval should be controlledaccurately as well. Furthermore, light emitted from each OIZ isseparated out from each other using a collection and separation opticssystem and detected by a different set of filters and photodetectors.The efficiency of light collected and delivered to photodetectors foreach OIZ depends on the separation distances between OIZs. If theseparation distance between these OIZs varies due to any factors such astemperature, pressure, misalignment during instrument shipping, or othersystem instability factors, it may affect not only the time intervalbetween detected pulses generated in two OIZs but also, moreimportantly, the efficiency of light collection for each OIZ, leading tounreliable measurement results of the signals. (3) The optical setup isnot efficient because each photodetector is used to detect only one typeof fluorescent molecule (i.e. having a pre-determined excitationwavelength and emission bandwidth). For example, in a system asillustrated in FIG. 1, four detection channels (E1 to E4) are needed torecord the signals from four fluorescent molecules (FM1 to FM4). If FM2(e.g. PE-Cy7) and FM4 (e.g. APC-Cy7) are assumed to have thesame/overlapped emission spectrum but need to be excited by S1 and S2respectively, two sets of band-pass filters and photodetectors are stillneeded for effective detection. Combining of the detection of FM2 andFM4 with one band-pass filter and one photodetector is not possible inthis configuration. This results in the increased complexity andtherefore increased cost of the whole system.

In the second approach, the shaped, elliptical laser beams from multiplesources (e.g., S1, S2 in FIG. 2) of different wavelengths propagateacross the flow channel at a single vertical location along the flowchannel, forming a single optical interrogation zone (OIZ). Thus when aparticle/cell flows through the flow channel, it will pass the OIZ andwill be subjected to multiple laser beams simultaneously. Consequently,light in laser beams will be scattered and fluorescent light will beemitted by fluorescent molecules (FM) possessed by the cell. Multiplefluorescent signals with same/overlapped or different emission peakwavelengths may be emitted by different fluorescent molecules excited bymultiple laser beams at the OIZ. For example, S1 and S2 can be 488 nmand 640 nm laser, respectively. Fluorescent molecules FITC and PE-Cy7can be excited by S1 (488 nm) and emit light at peak wavelength of 519nm and 785 nm, respectively, whilst fluorescent molecules APC andAPC-Cy7 can be excited by S2 (640 nm) and emit light at peak wavelengthof 660 nm and 785 respectively. Through light collection optics, thefluorescent signals emitted from the particle/cell in the OIZ would becollected, and then further split or filtered into different wavelengthranges and is detected by photodetectors, converting optical signalsinto electronic signals. For example, fluorescent signals emitted fromOIZ is split and detected by D1, D2 and D3 at three different emissionwavelengths to resolve quantity of fluorescent molecules FM1 (e.g.,FITC) and FM2 (e.g., PE-Cy7 or PE-Cy7) and FM3 (APC). Using electronicprocessors, electronic signals are then filtered, amplified andconverted to digital signals, and further processed to derive variouscharacteristics of each corresponding fluorescent molecule. For example,output E1, E2 and E3 correspond to the signals of FM1, FM2 and FM3,respectively. This second approach, as schematically represented in FIG.2, has some advantages. There is no need for complex optical system forseparating fluorescent signals excited by multiple light sources.Furthermore, the optical setup in this approach is efficient since thesame set of optical filter and photodetector could be used for detectionof fluorescent molecules having the same emission peak wavelengths butdifferent excitation wavelengths. For example, whilst PE-Cy7 and APC-Cy7are excited by 488 nm and 640 nm respectively, both molecules can bedetected using the same set of band-pass filter and one photodetectorfor monitoring wavelength ranges centered at 785 nm, as long as thesetwo dyes are not used in the same experiment. On the other hand, thisapproach has a major limitation that it could not distinguish thefluorescent signals from different fluorescent molecules having the sameemission spectra even if they are excited by different lasers. Forexample, the system of such a configuration as schematically shown inFIG. 2 could not be used to distinguish and reliably detect fluorescentsignals from molecules of PE-Cy7 (excited by 488 nm laser) and APC-Cy7(excited by 640 nm laser) in the same experiment, even though they havedifferent excitation wavelengths.

A relatively recent method, published in U.S. Pat. No. 7,990,525,describes an extension of this second approach where the excitationlaser light is time-multiplexed so that each light source is switched onand off at a very fast rate. At any time moment, no two (or more) lightsources are switched on simultaneously. Thus, either no light source oronly one light source is switched on by appropriate control of lightsources. The detection electronics can be used in synchronization sothat the fluorescence signals excited by different lasers could beisolated, recovered, and analyzed. It is required that the multipleexcitation light beams are directed/focused to the same OIZ, andconsequently there is no time interval between detected electronicsignals excited by different excitation light sources as seen for thefirst approach. However, this approach possessed other limitations.Firstly, in order to eliminate the above-mentioned time interval issue,it requires precise combination and alignment of the light beams fromdifferent excitation light sources to be coaxial and overlapped at thesame location across the flow channel. Secondly, for reliable recoveryand isolation of the emitted fluorescent signals excited by differentexcitation light sources as a particle/cell passes through the OIZ,accurate control of the time-multiplexed illumination of multipleexcitation light beams is essential as well as the subsequentsynchronization of the signal processing electronics. Thirdly, thetime-multiplexed illumination of multiple excitation light sourcescorresponds to a fact that the time interval for a cell/particle to passthrough the OIZ is shared between multiple excitations. Consequently, asingle particle-induced electronic pulse generated when theparticle/cell passes through the OIZ is time-shared between multipleexcitations as well. If the same amount of data points is needed toeffectively recover such particle-induced electronic pulse informationfor each of the multiple excitations, a faster multiplexing illuminationrate and a faster sampling frequency for the signal processingelectronics is required. Furthermore, such issue would become moresevere especially when the number of the excitation light sourcesincreases. Fourthly, such configuration requires that one excitationlight source is OFF when another one is ON. However, due to straycurrent of the electronic signal for the digital modulation of the lasersource and/or the property of the laser source (i.e. modulation ratio isnot high enough so that there is still low level of light from the lightsource even when it is controlled to be OFF), such OFF-status lightsource will still contribute some illumination to the OIZ thus increasethe background for detection and measurement of the emitted fluorescentsignal excited by another ON-status light source, affecting the systemsensitivity for detecting low-level, dim fluorescent particles/cells.

Another recent method, published in U.S. Pat. No. 8,077,310, alsodescribes a further extension of the previously described secondapproach where two excitation sources emit lights at differentwavelengths onto a single location on a flow channel. The multipleexcitation sources are controlled to operate between such operationalmodes: a first mode wherein only one of multiple excitation sourcesemits light onto the single location and a second mode wherein bothexcitation sources emit lights onto the single location. The approachfurther comprises a detector subsystem that detects lights emitted fromthe single location and generates a composite signal and a processor toseparate the composite signal into component signals due to each of twoexcitation sources. Since the multiple excitation light beams aredirected to the same single location on the flow channel, consequentlythere is no time interval between detected electronic signals excited bydifferent excitation sources as seen for the first approach. Byswitching between different operational modes, composite signalscorresponding to these operation modes are generated and can beprocessed to result in isolated signals due to each individualexcitation sources. This approach provides a possibility of, in a singleexperiment, using different fluorescent molecules having the sameemission spectra but with different excitation wavelengths. However,such an approach has limitations due to emitting multiple excitationsources onto a single location on the flow cell and simultaneous turn-onof multiple excitation sources at some time moments.

Still other approaches have been suggested or described in recent years,relating to emitting multiple excitation light sources onto a flowchannel and detecting and separating emission fluorescent lights due tothese excitation sources. For example, US 2008/0213915 described anapproach where multiple excitation light sources are all modulated witheach source being modulated at different frequencies. The modulatedexcitation beams are combined and guided onto single or multiple focusedspots or locations on the flow channel. The fluorescent emissions fromparticles due to modulated excitation beams are detected to producedetector output signals, which are then processed to distinguish thefluorescent signals caused by each individual excitation beam. Inanother example, US 2007/0096039 described an approach for analyzingobjects having multiple fluorescing species in a fluid stream. Multipleintensity-modulated excitation light beams, each of which is modulatedat a unique frequency between 2 and 100 MHz, are combined and directedto one or more interrogation zones on a flow cell and will interact withthe passing objects in a fluid stream in the flow cell. The fluorescenceemission light from fluorescent species in the objects is detected withone or more photosensitive detectors and resulted electronic signals areanalyzed to extract multiple component emission signals, each of whichcorresponds to one excitation light beam. These approaches havelimitations associated with the requirement of modulation of allexcitation sources at unique frequencies and the ineffectiveness in thede-modulation methods in achieving high signal-noise ratios. Thus, thereremains a need to develop a novel approach for effective detection ofemission light by multiple excitation light sources from a flow channel.

SUMMARY OF THE INVENTION

In one aspect of the invention, a system for detecting signal componentsof light induced by multiple excitation sources is provided, whichincludes: a flow channel configured for the flow of particles, the flowchannel including at least two spatially separated optical interrogationzones; a non-modulating excitation source that directs a light beam of afirst wavelength at a near constant intensity onto a first of theoptical interrogation zones; a modulating excitation source that directsa light beam of a second wavelength with an intensity modulated overtime at a modulating frequency onto a second of the opticalinterrogation zones, wherein the second wavelength is different from thefirst wavelength; a detector subsystem comprising a set of detectorsconfigured to detect light emitted from particles flowing through the atleast two optical interrogation zones and to convert the detected lightinto a total electrical signal; and a processor configured to receivethe total electrical signal from the detector subsystem, to de-modulateelectrical signal that is modulated, and to determine signal componentsfrom the light detected from each of the optical interrogation zones.

In some embodiments, the system further includes a third opticalinterrogation zone spatially separated from the first and second opticalinterrogation zones; and another excitation source that directs a lightbeam onto the third optical interrogation zone. In one example of suchembodiment, the excitation source that directs the light beam onto thethird optical interrogation zone is a second non-modulating excitationsource that directs the light beam of a third wavelength at a nearconstant intensity onto the third optical interrogation zone, whereinthe third wavelength is different from both the first wavelength and thesecond wavelength. In another example of such an embodiment, theexcitation source that directs the light beam onto the third opticalinterrogation zone is a second modulating excitation source that directsthe light beam of a third wavelength with an intensity modulated overtime at a modulating frequency.

In another aspect of the present invention, a method of detecting signalcomponents from light induced by multiple excitation sources isprovided, the method including: providing a flow channel including atleast two spatially separated optical interrogation zones; flowing apopulation of particles labeled with at least two different fluorescentmolecules through each of the optical interrogation zones; directing alight beam of a first wavelength at a near constant intensity onto afirst of the optical interrogation zones to induce emission of lightfrom the fluorescence-molecule containing particles; directing a lightbeam of a first wavelength with an intensity modulated over timeaccording to a modulating frequency onto a second of the opticalinterrogation zones to induce emission of light from thefluorescence-molecule containing particles, wherein the secondwavelength is different from the first wavelength; detecting the lightemitted from the particles from each of the optical interrogation zoneand converting detected light into a total electrical signal;de-modulating electrical signal from the total electrical; anddetermining signal components of the light detected from each of theoptical interrogation zones.

In some embodiments the method includes flowing the population ofparticles through a third optical interrogation zone spatially separatedfrom the first and second optical interrogation zones; directing a lightbeam of a third wavelength at a near constant intensity onto the thirdoptical interrogation zone to induce emission of light from thefluorescence-molecule containing particles, wherein the third wavelengthis different from the first and second wavelengths; detecting the lightemitted from the particles flowing through the third opticalinterrogation zone and converting the detected light into the totalelectrical signal.

In a related embodiment, the method includes flowing the population ofparticles through a third optical interrogation zone spatially separatedfrom the first and second optical interrogation zones; directing a lightbeam of a third wavelength with an intensity being modulated over timeat the modulating frequency onto the third optical interrogation zone toinduce emission of light from the fluorescence-molecule containingparticles, wherein the third wavelength is different the first andsecond wavelengths; and detecting the light emitted from the particlesflowing through the third optical interrogation zone and converting thedetected light into the total electrical signal.

For the present invention, multiple excitation light sources cancomprise at least 2 light sources of different wavelengths. In oneembodiment of the invention, the multiple light sources comprise 2 lightsources having different wavelengths. During operation, one light sourceis not intensity modulated and one light source is intensity-modulated.In another embodiment of the invention, the multiple light sourcescomprise 3 light sources having different wavelengths. During operation,at least one light source is not modulated and at least one light sourceis intensity-modulated. For example, in one embodiment, one light sourceis intensity-modulated and the other two light sources are notmodulated. For such an embodiment, the light beams from the three lightsources may be arranged along the flow cell in such an order that themodulated light beam is positioned in the middle with the twoun-modulated light beams positioned each at one side (or end) of themodulated beam along the flow cell. In another embodiment, one lightsource is not modulated and the other two light sources areintensity-modulated. For such an embodiment, the light beams from thethree light sources may be arranged along the flow cell in such an orderthat the un-modulated light beam is positioned in the middle with thetwo modulated light beams positioned each at one side (or end) of theun-modulated beam along the flow cell. The modulation frequencies of twointensity-modulated light sources may be different. In certain preferredembodiment, the modulation frequencies of two intensity-modulated lightsources are the same. In still another embodiment of the invention, themultiple light sources comprise 4 light sources having differentwavelengths. During operation, at least one light source is notmodulated and at least one light source is intensity-modulated. Forexample, in one embodiment, two light sources are intensity-modulatedand the other two light sources are not modulated. For such anembodiment, the light beams from the four light sources may be arrangedalong the flow cell in such an order that the un-modulated light beamand modulated light team are positioned in an alternative manner. Themodulation frequencies of two intensity-modulated light sources may bedifferent. In certain preferred embodiment, the modulation frequenciesof two intensity-modulated light sources are the same. In still anotherembodiment of the invention, the multiple light sources comprise morethan 4 light sources having different wavelengths. A light source couldbe a laser, a laser emitting diode (LED) or other light generatingcomponent. At least one of the multiple light sources can be modulatedand at least one of the multiple light sources is not modulated and thusmaintains a constant or near constant intensity. For N light sources (Nis the total number of different excitation light sources with differentwavelength), some light sources can be modulated and other light sourcesare not-modulated. Preferably, light beams from these light sources maybe arranged along the flow cell in such an order that the un-modulatedlight beam and modulated light team are positioned in an alternativemanner. The modulation frequencies of those intensity-modulated lightsources may be different. In certain preferred embodiments, themodulation frequencies of the intensity-modulated light sources are thesame. Light sources can be either analogue modulated or digitallymodulated. In analogue modulation, light intensity from the light sourcecan be controlled to be proportional to the amplitude of the analoguesignals used for analogue modulation. The analogue signals used formodulation can be of different waveforms, including sine-waveform,triangular-waveform, and seesaw-waveform. In digital modulation, lightfrom the light source can be turned on and off by the digital signalsused for digital modulation. The light source can be modulated at anysuitable frequencies, as long as the modulated particle-induced (orcell-induced) electronic pulses can be de-modulated throughelectronics-processing means to recover the particle-induced electronicpulse.

Each optical interrogation zone corresponds to a region where the lightbeam from an excitation light source is propagating across the flowchannel. The light beam typically has an “elliptical” or “rectangular”shape with the major/long axis perpendicular to the flowing direction ofthe particles/cells in a flow channel and minor/short axis parallel tothe flowing direction of the particles/cells in a flow channel. In oneembodiment, the minor/short axis of the light beams is between about 5and 30 microns and the major/long axis of the light beam is between 50and 200 microns. Preferably, the minor/short axis of the beam is between10 and 20 microns. Preferably, the major/long axis of the beam isbetween 60 and 100 microns. The light illumination sub-system of thepresent invention shall include beam-shaping optical components to shapethe beam to the desired shape and power distribution.

In the present invention, the optical interrogation zones may beseparated from each other and arranged to be at different locationsalong the flow channel such that a cell or particle flows through aseries of optical interrogation zones with the center-to-center distancebetween adjacent optical interrogation zones of different values. In oneembodiment, the excitation beam has a elliptical shape whose minor axisis 10 microns. The center-to-center distance between adjacent OIZs couldbe as large as more than 500 microns. Preferably, the center-to-centerdistance between adjacent OIZs is between 10 microns and 300 microns.Even more preferably, the center-to-center distance between adjacentOIZs is between 20 microns and 200 microns. Still more preferably, thecenter-to-center distance between adjacent OIZs is between 25 micronsand 100 microns. Still more preferably, the center-to-center distancebetween adjacent optical interrogation zones is between 30 microns and80 microns. Still more preferably, the center-to-center distance betweenadjacent optical interrogation zones is between 35 microns and 70microns. The choice of the center-to-center distance between adjacentOIZs should be dependent on a number of factors, including the minoraxis of light beam along the flow direction at OIZs, the lightcollection efficiency for light emitted from different OIZs, the flowspeed range of particles/cells flowing inside the flow channel and theparticle/cell concentration range. Preferably, the light beams atadjacent OIZs do not overlap. For example, if the excitation beam has anelliptical shape whose minor axis is 15 microns parallel to the flowdirection, the center-to-center distance between adjacent OIZs ispreferably between 15 microns and 150 microns, leaving the gap betweenthe adjacent light beams propagating through the flow channel in therange of 0 to 135 microns. In one embodiment, if the center-to-centerdistance is 30 microns for the beams with 15 micron in minor axis beams,the gap between the adjacent light beams is 15 microns. The lightillumination sub-system of the present invention shall includebeam-steering and/or beam combining and/or light focusing and/or otheroptical components to deliver excitation light beams with required beamshape to different locations with required center-to-center distancealong the flow channel.

A detection sub-system includes light collection optics capable ofcollecting light emitted from different optical interrogation zones. Thecollected light is then split or filtered into light of interest withdifferent wavelength ranges and is detected via photodetectors such asPMT (photo-multiplier tubes), APD (avalanche photodiodes) and the like.In the present invention, the emitted light from all the OIZs iscollected and then preferably filtered by band-pass filters to split allthe collected light into different wavelength ranges. Then a set ofcorresponding photodetectors convert the light signals into electronicsignals and such electronic signals are processed by an electronicsprocessor using methods including amplification, multiplying with asignal, filtering, de-modulation, A/D conversion, signal recovery means,etc. Since at least one excitation light source in the present inventionmay be modulated, the resulting electronics signal, i.e.particle-induced (or cell-induced) pulse from the photodetector would becorrespondingly modulated as well. De-modulation andsignal-recovery-means are used to recover these particle-induced pulsesand correlate each individual particle-induced pulse to its excitationwavelength. Different de-modulation and signal-recovery-means can beemployed, including single-side demodulation, quadrature demodulationand square-wave demodulation. In this way, the filter and photodetectorset for detecting the same emission wavelength range can be shared eventhough the emission light may be excited by different excitation lightsources. Therefore, it shows advantages over the first approach in theprior art, as schematically represented in FIG. 1. In addition, there isno strict requirement for the location of optical interrogation zonesfor effective detection using present invention, as long as theseparation distance of the adjacent OIZs are properly selected so thatthe probability of two particles/cells being both within two OIZs isminimized or very low. Therefore, the change of the separation distanceof the OIZs caused by any factors such as temperature, pressure,misalignment during instrument shipping, or other system instabilityfactors, will not affect the measurement results of the signals.Furthermore, as an advantage over the time-multiplexed illumination ofmultiple excitation light beams in U.S. Pat. No. 7,990,525, the systemand method in the present invention do not need for accuratesynchronization between the modulations of each excitation light source(i.e., the modulation of each excitation light source is independent ofeach other).

In addition, compared with the approaches described in U.S. Pat. No.8,077,310 where light from different excitation sources emit to the samelocation on the flow cell, the system and method in the presentinvention directs light from different excitation sources onto differentlocations, allowing for the use of full dynamic range of photodetectorsfor detecting the fluorescent signals caused by each individual lightexcitation source and minimizing possible interference or crosstalkbetween fluorescent signals caused by different excitation sources.

Finally, compared with approaches described in US 2007/0096039 and US2008/0213915 where multiple excitation light sources are all modulatedwith each source being modulated at different frequencies, the systemand method in the present invention require that at least one lightsource is not modulated. In addition, when multiple light sources areintensity-modulated, their modulation frequencies are preferably to bethe same. These approaches offer significant advantages in reducing thecomplexity of electronic-processor sub-systems and in improving thesystem performance such as signal-to-noise ratio, signal process flow aswell as data analysis and process speed.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic representation of a prior flow cytometry lightillumination approach with two excitation light sources. Light beams arearranged or directed to two optical interrogation zones, separated at afixed distance along the flow channel. Light emitted from different OIZsis collected and optically/physically separated. Separated lightcomponents due to different excitation sources are then split orfiltered into light components of different wavelength ranges and aredetected by photodetectors. The electronic signals from photodetectorsare processed using electronics processor. Each electronic signal outputcorresponds to light emitted from one optical interrogation zone at aparticular wavelength range due to one excitation source.

FIG. 2 is a schematic representation of a prior flow cytometry lightillumination approach with two excitation light sources. Light beamsfrom the two excitation light sources are directed to a single opticalinterrogation zone (OIZ). Light emitted from the OIZ is collected andthen split or filtered into light components of different wavelengthranges and are detected by photodetectors. The electronic signals fromphotodetectors are processed using electronics processor. Electronicsignal output corresponds to light emitted from the opticalinterrogation zone at a particular wavelength range due to either one ofthe light source or both.

FIG. 3A shows an exemplary schematic representation of the presentinvention where a light illumination sub-system directs light beams fromtwo excitation light sources S1, S2 to two optical interrogation zonesOIZ1, OIZ2, separated at some distances along the flow channel. FIG. 3Bshows an exemplary schematic representation of the present inventionwhere a light illumination sub-system directs light beams from twoexcitation light sources S1, S2, S3 to three optical interrogation zonesOIZ1, OIZ2, OIZ3, separated at some distances along the flow channel.Light emitted from the two OIZs (FIG. 3A) or three OIZs (FIG. 3B) iscollected using same light collection optics. The collected light isthen split or filtered into light components of different wavelengthranges and is detected by a set of photodetectors. The electronicsignals from photodetectors are processed using electronics processor.FIG. 3A depicts a preferred embodiment, where one light source S2 ismodulated and the other S1 is not modulated. FIG. 3B depicts a preferredembodiment, where two light sources S2 are modulated and another lightsource S1 is not modulated. Using de-modulation and signal-recovermeans, electronics processor can de-modulate the electronic signals fromthe photodetectors to recover particle-induced electronic pulse due toeither light source.

FIGS. 4A-H show the process of analogue modulation of light sources andde-modulation of electronic signals to recover particle-inducedelectronic pulse, as a particle/cell passes through two opticalinterrogation zones: FIG. 4A) an analogue signal used to modulate the1^(st) light source (3 MHz); FIG. 4B) an analogue signal used tomodulate the 2^(nd) light source (6 MHz); FIG. 4C) detected electronicsignals if no modulation of either light source is employed where the1^(st) and 2^(nd) pulse is generated as a particle/cell passes throughthe 1^(st) OIZ and the 2^(nd) OIZ, respectively; FIG. 4D) detectedelectronic signals when analogue modulations shown in FIG. 4A) and FIG.4B) are applied to the 1^(st) light source and the 2^(nd) light source,respectively; FIG. 4E) the intermediate de-modulation signal used torecover the 1^(st) particle-induced electronic pulse; FIG. 4F) theintermediate de-modulation signal used to recover the 2^(nd)particle-induced electronic pulse; FIG. 4G) recovered 1^(st) pulseprofile; and FIG. 4H) recovered 2^(nd) pulse profile.

FIGS. 5A-F shows the process of analogue modulation of light sources andde-modulation of electronic signals to recover particle-inducedelectronic pulse, as a particle/cell passes through two opticalinterrogation zones: FIG. 5A) an analogue signal used to modulate the1^(st) light source (3 MHz); FIG. 5B) detected electronic signals if nomodulation of the 1^(st) light source is employed where the 1^(st) and2^(nd) pulse is generated as a particle/cell passes through the 1^(st)OIZ and the 2^(nd) OIZ, respectively; FIG. 5C) detected electronicsignals when analogue modulations shown in FIG. 5A) is applied to the1^(st) light source; FIG. 5D) the intermediate de-modulation signal usedto recover the 1^(st) particle-induced electronic pulse by multiplyingthe detected electronic signals in FIG. 5C) with the modulationsine-wave signal; FIG. 5E) recovered 1^(st) pulse profile by low-passfiltering the intermediate de-modulation signal in FIG. 5D); and FIG.5F) recovered 2^(nd) pulse profile by low-pass filtering the detectedelectronic signals in FIG. 5C).

FIGS. 6A-I shows the process of analogue modulation of light sources andde-modulation of electronic signals to recover particle-inducedelectronic pulse, as a particle/cell passes through three opticalinterrogation zones: FIG. 6A) an analogue signal used to modulate the1^(st) light source (3 MHz); FIG. 6B) an analogue signal used tomodulate the 3rd light source (6 MHz); FIG. 6C) detected electronicsignals if no modulation of light sources is employed where the 1st,2^(nd) and 3^(rd) pulse is generated as a particle/cell passes throughthe 1^(st) OIZ, the 2^(nd) OIZ and the 3^(rd) OIZ, respectively; FIG.6D) detected electronic signals when analogue modulations shown in (a)and (b) are applied to the 1^(st) light source and the 3^(rd) lightsource, respectively; FIG. 6E) the intermediate de-modulation signalused to recover the 1^(st) particle-induced electronic pulse bymultiplying the detected electronic signals in (d) with the modulationsine-wave signal in FIG. 6A); FIG. 6F) the intermediate de-modulationsignal used to recover the 3^(rd) particle-induced electronic pulse bymultiplying the detected electronic signals in FIG. 6D) with themodulation sine-wave signal in FIG. 6B); FIG. 6G) recovered the 1^(st)pulse profiles by low-pass filtering the intermediate de-modulationsignal in FIG. 6E); FIG. 6H) recovered the 2^(nd) pulse profile bylow-pass filtering the detected electronic signal in FIG. 6D); and FIG.6I) recovered the 3^(rd) pulse profile by low-pass filtering theintermediate de-modulation signal in FIG. 6F).

FIGS. 7A-H shows the process of analogue modulation of light sources andde-modulation of electronic signals to recover particle-inducedelectronic pulse, as a particle/cell passes through three opticalinterrogation zones: FIG. 7A) an analogue signal used to modulate the1^(st) and the 3^(rd) light source (3 MHz); FIG. 7B) detected electronicsignals if no modulation of light sources is employed where the 1^(st),2^(nd) and 3^(rd) pulse is generated as a particle/cell passes throughthe 1^(st) OIZ, the 2^(nd) OIZ and the 3^(rd) OIZ, respectively; FIG.7C) detected electronic signals when analogue modulation shown in FIG.7A) is applied to both the 1^(st) light source and the 3^(rd) lightsource; FIG. 7D) the intermediate de-modulation signal used to recoverthe 1^(st) and 3^(rd) particle-induced electronic pulse by multiplyingthe detected electronic signals in FIG. 7C) with the modulationsine-wave signal in FIG. 7A); FIG. 7E) recovered 1^(st) and 3^(rd) pulseprofile by low-pass filtering the intermediate de-modulation signal inFIG. 7D); FIG. 7F) recovered 1^(st) pulse profile through identifyingtime-window corresponding to particle passing through the 1^(st) OIZ;FIG. 7G) recovered 2^(nd) pulse profile by low-pass filtering thedetected electronic signals in FIG. 7C); and FIG. 7H) recovered 3^(rd)pulse profile through identifying time-window corresponding to particlepassing through the 3^(rd) OIZ. Note that in this example, the samemodulation signal is used for modulating the 1^(st) and 3^(rd) lightsources. It is possible to separate, isolate and identify the 1^(st) and3^(rd) pulse profiles caused by particle traveling through the 1^(st)OIZ and the 3^(rd) OIZ, based on identifying appropriate time windows.

DESCRIPTION OF PREFERRED EMBODIMENTS

In one aspect of the invention, a system for detecting signal componentsof light induced by multiple excitation sources, which includes a flowchannel configured for the flow of particles, the flow channel includingat least two spatially separated optical interrogation zones; anon-modulating excitation source that directs a light beam of a firstwavelength at a near constant intensity onto a first of the opticalinterrogation zones; a modulating excitation source that directs a lightbeam of a second wavelength with an intensity modulated over time at amodulating frequency onto a second of the optical interrogation zones,wherein the second wavelength is different from the first wavelength; adetector subsystem comprising a set of detectors configured to detectlight emitted from particles flowing through the at least two opticalinterrogation zones and to convert the detected light into a totalelectrical signal; and a processor configured to receive the totalelectrical signal from the detector subsystem, to de-modulate electricalsignal that is modulated, and to determine signal components from thelight detected from each of the optical interrogation zones.

In an embodiment of the system of the present invention, the systemfurther comprises a third optical interrogation zone spatially separatedfrom the first and second optical interrogation zones; and anotherexcitation source that directs a light beam onto the third opticalinterrogation zone. In one example of such embodiment, the excitationsource that directs the light beam onto the third optical interrogationzone is a second non-modulating excitation source that directs the lightbeam of a third wavelength at a near constant intensity onto the thirdoptical interrogation zone, wherein the third wavelength is differentfrom both the first wavelength and the second wavelength. In anotherexample of such an embodiment, the excitation source that directs thelight beam onto the third optical interrogation zone is a secondmodulating excitation source that directs the light beam of a thirdwavelength with an intensity modulated over time at a modulatingfrequency.

To this end, FIG. 3 describes a preferred embodiment of a flow cytometersystem, comprising a light illumination sub-system comprising multiplelight sources, at least one of which is intensity-modulated and at leastone of which is not modulated and thus provides a constant or nearconstant intensity, to form multiple optical interrogation zones (OIZs)at different locations along a flow channel, a detection sub-system thatdetects light emitted from multiple OIZs including light collectionoptics, light splitters/filters, and photodetectors, an electronicprocessor sub-system connected to the detection sub-system and capableof isolating and recovering the signal components due to differentexcitation sources. For preferred embodiments of the present invention,each excitation light source emits light into a single OIZ and differentexcitation light sources will emit lights into different OIZs.

Preferably, each light source emits a light beam of a different lightwavelength to one of at least two, three or more optical interrogationzones. The light wavelengths can be of any values, as long as they areavailable for exciting fluorescent molecules to be detected for a flowcytometry application. The wavelengths may be selected from rangescentered at about 325 nm, 355 nm, 365 nm, 375 nm, 405 nm, 407 nm, 445nm, 458 nm, 460 nm, 480 nm, 488 nm, 514 nm, 532 nm, 552 nm, 561 nm, 568nm, 577 nm, 595 nm, 633 nm, 635 nm, 640 nm, 647 nm, 660 nm, 685 nm, orthe like. The term “ranges centered at about” is intended to encompassother neighboring wavelengths as desired, such as about +/−5 nm or asknown in the flow cytometry arts.

The light source may be different power levels from as low as a coupleof milli-watts to as large as a thousand milli-watts. Preferably, thepower of light sources may vary between 2 mW and 1000 mW. Morepreferably, the power of light sources may vary between 5 mW and 500 mW.Still more preferably, the power of light sources may vary between 10 mWand 200 mW. Even more preferably, the power of light sources may varybetween 15 mW and 100 mW. Preferably at least one light beam used forthe excitation of fluorescence is intensity modulated and preferably atleast one light beam used for the excitation of fluorescence maintains anear constant intensity. By “intensity modulated” it is meant that theintensity (or the power) of the light beam is modulated at a modulatingfrequency. Approaches to modulate intensity according to a modulatingfrequency are discussed in detail in sections that follow. In contrast,a beam that maintains a “near constant intensity” refers to a beam thatis not modulated and thus retains its intensity or its power withoutsignificant deviation throughout the detection or measurement process.

The detector subsystem of the present invention includes a set ofdetectors for selectively detecting any appropriate wavelengths that areemitted by fluorescent molecules of interest. For instance, a detectorsubsystem may detect fluorescent wavelength ranges centered at about 421nm, 450 nm, 455 nm, 519 nm, 530 nm, 578 nm, 585 nm, 603 nm, 615 nm, 620nm, 650 nm, 660 nm, 785 nm and the like. The examples of detectorwavelength ranges may include 421±30 nm, 450±30 nm, 455±40 nm, 519±30nm, 530±15 nm, 578±15 nm, 585±40 nm, 603±30 nm, 615±30 nm, 620±30nm, >650 nm, 660±10 nm, 667±30 nm, 668±30 nm, 678±30 nm, 695±25 nm, >750nm, 780±30 nm and >785 nm. The fluorescent molecules of interestinclude, but not limited to those offered under the names PACIFIC BLUE,BD HORIZON V450, DAPI, HOECHST BLUE, ALEXA FLUOR 450, Indo-1 Violet,VIOBLUE, CFP, CLICK-IT EdU PACIFIC BLUE, PO-PRO-1, DYECYLCE Violet,LIVE/DEAD Fixable Violet Dead Cell Stain, Calcein Violet, QDOT 525,AMCYAN, SYTOX Blue, PACIFIC ORANGE, QDOT 565, QDOT 585, LIVE/DEADFixable Aqua Dead Cell Stain, QDOT 605, QDOT 655, QDOT 800, Fluorescein,FITC, ALEXA FLUOR 488, QDOT 525, Calcein, FLUO-3 or FLUO-4, TO-PRO-1,CFSE, GFP, EGFP, EYFP, JC-1, DiOC₂(3), DiIC₂(3), SYNTOX Green, DYECYLCEGreen, Rhodamine 123, Rhodamine 110, LIVE/DEAD Fixable Green Dead CellStain, YO-PRO-1, Indo-1 Blue, CY-2, Acridine Orange, PKH2, BCECF, PI,RPE, ALEXA FLUOR 546, PE-CY5, LP Hoechst Red, PERCP-CY7, FURA RED,DECYCLE Orange, JC-1, DiOC₂(3), SNARF (low pH), PHRODO dye, PE-TEXASRED, CY3, Pyronin Y, dsRED, PKH26, BCECF, Resorufin RPE-ALEXA FLUOR 610,PI, RPE-TEXAS RED, PI, JC-1, LIVE/DEAD Fixable Red Dead Cell Stain,RPE-ALEXA FLUOR 700, RPE-CY 5.5, TRICOLOR, PERCP, PI, 7-AAD, SNARF (highpH), APC, ALEXA FLUOR 647, ALEXA FLUOR 647, LIVE/DEAD Fixable Far RedDead Cell Stain, TO-PRO 3, SYNTOX Red, MITOPROBE DilC1(5), CELLTRACE FarRed DDAO-SE RPE-ALEXA FLUOR, ALEXA FLUOR 647, CLICK-IT EdU ALEXA FLUOR647, CY5, APC-CY5.5, DRAQ5APC-CY7, LIVE/DEAD Fixable Near-IR Dead CellStain.

The invention also provides a method of detecting signal components fromlight induced by multiple excitation sources. The method including:providing a flow channel including at least two spatially separatedoptical interrogation zones; flowing a population of particles labeledwith at least two different fluorescent molecules through each of theoptical interrogation zones; directing a light beam of a firstwavelength at a near constant intensity onto a first of the opticalinterrogation zones to induce emission of light from thefluorescence-molecule containing particles; directing a light beam of afirst wavelength with an intensity modulated over time according to amodulating frequency onto a second of the optical interrogation zones toinduce emission of light from the fluorescence-molecule containingparticles, wherein the second wavelength is different from the firstwavelength; detecting the light emitted from the particles from each ofthe optical interrogation zone and converting detected light into atotal electrical signal; de-modulating electrical signal from the totalelectrical; and determining signal components of the light detected fromeach of the optical interrogation zones.

In some embodiments the method includes flowing the population ofparticles through a third optical interrogation zone spatially separatedfrom the first and second optical interrogation zones; directing a lightbeam of a third wavelength at a near constant intensity onto the thirdoptical interrogation zone to induce emission of light from thefluorescence-molecule containing particles, wherein the third wavelengthis different from the first and second wavelengths; detecting the lightemitted from the particles flowing through the third opticalinterrogation zone and converting the detected light into the totalelectrical signal.

In a related embodiment, the method includes flowing the population ofparticles through a third optical interrogation zone spatially separatedfrom the first and second optical interrogation zones; directing a lightbeam of a third wavelength with an intensity being modulated over timeat the modulating frequency onto the third optical interrogation zone toinduce emission of light from the fluorescence-molecule containingparticles, wherein the third wavelength is different the first andsecond wavelengths; and detecting the light emitted from the particlesflowing through the third optical interrogation zone and converting thedetected light into the total electrical signal.

Thus an exemplary system of the present invention operates in the waydescribed as below. Fluorescent particles/cells are flown through theflow channel and pass through two optical interrogation zones at twodifferent locations along the flow channel. The shaped elliptical laserbeams from multiple sources (e.g., S1, S2 in FIG. 3) of differentwavelengths propagate across the flow channel and form two opticalinterrogation zones (OIZ1 and OIZ2) at different vertical locationsalong the flow channel. When a particle/cell flows through the flowchannel, it will pass the individual OIZ (e.g. OIZ1 corresponding tolaser beam S1 and OIZ2 corresponding to laser beam S2 in FIG. 3) insequence. Consequently, fluorescent light will be emitted in sequence aswell by different fluorescent molecules (FM) possessed by theparticle/cell. In prior art as shown in FIG. 1, light from two locationsis collected and further optically/physically separated to two differentpositions separated in a distance large enough to physically accommodateoptical filters and detectors in order to detect the signals. However,in the present invention, light from multiple locations (e.g. twolocations in FIG. 3) is collected using collection optics but does notneed to be further separated. Collected light is then split intodifferent components covering different wavelength ranges of the lightspectrum using splitters and filters. Each light component covering awavelength range is detected by a photodetector to output an electronicsignal. As particles/cells flow through in the flow channel, eachparticle/cell generates one light-scatter-induced electronic pulse andone or multiple fluorescence-induced electronic pulse dependent on thefluorochrome labeling of the particle/cell as it passes through eachOIZ.

If the emission light of the two different FMs excited by differentexcitation light source are not the same or do not overlap, thenfluorescent light emitted by these two FMs will be detected separatelyby two different photodetectors covering different wavelength ranges. Onthe other hand, if the emission spectra of two FMs are the same oroverlapped, the fluorescent light from the two types of FMs will bedetected by same photodetectors. The isolation/recovery of thefluorescent light component due to either type of FMs is achievedthrough modulation of light sources and de-modulation of the electronicsignals. In one example, both light sources are intensity-modulated witheach source modulated at a same unique frequency. In a preferredembodiment, one light source is intensity-modulated at a certainfrequency and the other light source is not modulated. Below we discussthese two exemplary approaches.

In one exemplary embodiment, for FIG. 3A, the 1^(st) excitation lightsource having the 1^(st) wavelength is modulated at one frequency whilstthe 2^(nd) light source having the 2^(nd) wavelength is modulated atanother frequency. Thus, resulting fluorescent signals (FS1 and FS2 inFIG. 3A) excited by the 1^(st) excitation light source and the 2^(nd)excitation light source as the particle/cell passes through the two OIZs(OIZ1 and OIZ2) and their corresponding electronic signals fromphotodetectors are also modulated at two corresponding frequencies.Modulated electronic signals due to each light source can bede-modulated separately to recover the pulse profile through certainmodulation-frequency-dependent electronic signal processing means. Thus,it is possible to isolate and detect fluorescent light from each type offluorescent signals excited by two different light sources. As anexample in FIG. 3A, S1 can be 488 nm laser and S2 can be 640 nm laser.Fluorescent molecules FITC and PE-Cy7 can be excited by S1 (488 nm) andemit light at peak wavelength of 519 nm and 785 nm respectively whilstAPC and APC-Cy7 can be excited by S2 (640 nm) and emit light at peakwavelength of 660 nm and 785 nm respectively. FS1 may comprisefluorescent signals from two fluorescent molecules FM1 (e.g., FITC) andFM2 (e.g., PE-Cy7) and FS2 may comprise fluorescent signals from twofluorescent molecules FM3 (e.g., FITC) and FM4 (e.g., PE-Cy7). Both FS1and FS2 are collected together through collection optics and are thensplit in three different wavelength ranges peaked at 519 nm (from FM1:FITC), 660 nm (from FM3: APC) and 785 nm (from FM2: PE-Cy7) and FM4:APC-Cy7) and detected by three photodetectors D1, D2 and D3,respectively. The electronic signals from photodetectors are processedvia electronics processor to derive four output signals E1, E2, E3 andE4, corresponding fluorescent signals form FM1, FM2, FM3 and FM4,respectively. Specifically, relating to the overlapped emission spectrafrom FM2 and FM4, the photo-detector D3 converts fluorescent signalspeaked at 785 nm from FM2 and FM4 into a combined electronic signal. Toseparate signal components, light sources S1 and S2 are modulated at twodifferent frequencies. Thus, the electronic signal componentscorresponding to FM2 and FM4 are modulated at different frequencies.Using the electronics processor, these modulated electronic signals arede-modulated to derive electronic signals corresponding to each type offluorescent molecules FM2 and FM4.

In yet a preferred embodiment, for FIG. 3A, the 1^(st) excitation lightsource S1 having the 1^(st) wavelength is not modulated whilst the2^(nd) light source S2 having the 2^(nd) wavelength is modulated at acertain frequency. Thus, resulting fluorescent signal FS1 and resultingscattered laser light signal in FIG. 3A excited by the 1^(st) excitationlight source as the particle/cell passes through OIZ1 and thecorresponding electronic signals from photodetectors are not intensitymodulated. Such non-modulated electronic signals would be in the form ofa pulse profile with time. On the other hand, resulting fluorescentsignal FS2 in FIG. 3A excited by the 2^(nd) excitation light source asthe particle/cell passes through OIZ2 and the corresponding electronicsignals from photodetectors are modulated at the same frequency as thatof modulation for the 2^(nd) light source. Such modulated electronicsignals due to the 2^(nd) light source can be de-modulated separately torecover the pulse profile through certain modulation-frequency-dependentelectronic signal processing means. Because of spatial separation ofOIZ1 and OIZ2, there is a time difference between the two pulseprofiles, one directly obtained from photodetectors due to the 1^(st)excitation light and another recovered through demodulation of themodulated electronic signals from photodetectors due to the 2^(nd)excitation light. Such time difference is dependent on spatial distanceof OIZ1 and OIZ2, and on the travel speed of particle/cell in the flowcell. Thus, it is possible to isolate and detect fluorescent light fromeach type of fluorescent signals excited by two different light sources.As an example in FIG. 3A, S1 can be 488 nm laser and S2 can be 640 nmlaser. Fluorescent molecules FITC and PE-Cy7 can be excited by S1 (488nm) and emit light at peak wavelength of 519 nm and 785 nm respectivelywhilst APC and APC-Cy7 can be excited by S2 (640 nm) and emit light atpeak wavelength of 660 nm and 785 nm respectively. FS1 may comprisefluorescent signals from two fluorescent molecules FM1 (e.g., FITC) andFM2 (e.g., PE-Cy7) and FS2 may comprise fluorescent signals from twofluorescent molecules FM3 (e.g., FITC) and FM4 (e.g., PE-Cy7). Both FS1and FS2 are collected together through collection optics and are thensplit in three different wavelength ranges peaked at 519 nm (from FM1:FITC), 660 nm (from FM3: APC) and 785 nm (from FM2: PE-Cy7) and FM4:APC-Cy7) and detected by a set of three photodetectors D1, D2 and D3,respectively. The electronic signals from photodetectors are processedvia electronics processor to derive four output signals E1, E2, E3 andE4, corresponding fluorescent signals form FM1, FM2, FM3 and FM4,respectively. Specifically, relating to the overlapped emission spectrafrom FM2 and FM4, the photo-detector D3 converts fluorescent signalspeaked at 785 nm from FM2 and FM4 into a combined electronic signal. Toseparate signal components, light sources S1 is not intensity modulatedand light source S2 is intensity modulated at a certain frequency. Thus,the electronic signal component corresponding to FM2 is not modulatedand can be obtained after straightforward signal processing such asamplification and low-pass filtering on the signals directly from thephotodetector. The electronic signal component corresponding to FM4 ismodulated at the frequency of modulation for light source S2 and suchmodulated electronic signals are de-modulated to derive electronicsignals corresponding to fluorescent molecule FM4. Examples ofde-modulation approaches will be described in the following sections.

There are several advantages of this preferred embodiment where onelight source is not modulated and another light source is modulated,comparing to the case of both light sources are modulated. First, asparticle/cell passes through OIZ1 where the 1^(st) light source is notmodulated, the particle scattered light signals (both forward scatterand side scatter signals) are also not modulated and would be in theform of a pulse-profile. Such particle scattered pulse profiles can bedirectly-used for determining appropriate time window for correspondingfluorescent signals due to the 1^(st) light source, and for determiningappropriate time windows for fluorescent signals due to the 2^(nd) lightsource after taking into account of the spatial distance between OIZ1and OIZ2 and the travel speed of particle/cell in the flow cell. Use ofsuch particle-scattering signals as a time window for fluorescentsignals is beneficial to filter out unwanted noises in fluorescentdetection channels. Also, particle scattering signals depends onprimarily on particle size, shape and surface scattering properties anddo not rely on presence of fluorescent molecules in the particle. Thus,particle scattering signals can be reliably obtained even if theparticle fluorescent signals are low. If the 1^(st) light source ismodulated, then such particle scattered signals would also be modulated.The modulated scatter signals could not be used directed for determiningthe corresponding time window for fluorescent signals. Thus, one wouldneed to first de-modulated the modulated scatted signals to recover thescatter-signal pulse profile. Such approach would lead to complexprocedures in processing various signals. It's much more preferred tohave 1^(st) light source un-modulated and use its corresponding scattedsignal to determine fluorescent signal windows. Secondly, fluorescentsignals and corresponding electronic signals due to the 1^(st) lightsource are not modulated, avoiding the need of de-modulation steps orprocesses for recovering the corresponding electronic pulse profiles.This is beneficial to reducing over system complexity involved in signalprocessing. Without the need for demodulation of fluorescent signals andscattered signals due to the 1^(st) light sources, more system resourcescould be employed for processing fluorescent signals due to the 2^(nd)light source, potentially improving signal to noise ratio for suchchannels. Thirdly, since one light source is modulated, choice ofsuitable modulation frequency would be more straightforward, comparedwith the case where two light sources are modulated at two differentmodulation frequencies.

The light illumination sub-system comprises multiple light sources usedto excite fluorescent molecules (FM) when a particle/cell flows throughthe flow channel. In the example of FIG. 3A, two excitation lightsources S1, S2 having different wavelengths are used. During operation,it is preferred that one light source S1 is not modulated and thusmaintains a constant intensity at the flow channel and one light sourceS2 is intensity-modulated.

In another embodiment of the invention, three excitation light sourceshaving different wavelengths can be used. In a preferred embodiment, atleast one light source is not modulated and at least one light source isintensity-modulated. For example, in one embodiment, one light source isintensity-modulated and the other two light sources are not modulated.For such an embodiment, the light beams from the three light sources maybe arranged along the flow cell in such an order that the modulatedlight beam is positioned in the middle with the two un-modulated lightbeams positioned each at one side (or end) of the modulated bean alongthe flow cell. As shown in FIG. 3B, in another embodiment, one lightsource S1 is not modulated and the other two light sources S2, S3 areintensity-modulated. For such an embodiment, the light beams from thethree light sources S1, S2, S3 may be arranged along the flow cell insuch an order that the un-modulated light beam is positioned in themiddle with the two modulated light beams positioned each at one side(or end) of the un-modulated beam along the flow cell. The modulationfrequencies of two intensity-modulated light sources S2, S3 may bedifferent. In preferred embodiments, the modulation frequencies of twointensity-modulated light sources S2, S3 are the same. Since there arespatial distances between light beams from the three different lightsources S1, S2, S3 along the flow direction of the flow cell,fluorescent signals caused by each light source S1, S2, S3 would occurat different time windows as particle/cell travel through threedifferent optical interrogation zones OIZ1, OIZ2, OIZ3. By takingadvantage of such time differences among fluorescent signal pulseprofiles due to different light sources, two of the modulated lightsources S2, S3 may employ the same modulation frequency. This way, thesame modulation signals can be used to modulate both light sources S2,S3, simplifying the system design. Furthermore, the same de-modulationprocessor or circuits can be used to de-modulate thefluorescence-corresponding electronic signals from both modulated lightsources S2, S3. This would significantly reduce the systemhardware/software complexity and improve system reliability andperformance. Thus, there are important benefits and advantages withusing a same modulation frequency for light sources being modulated,compared with the embodiments where different modulation frequencies areused for the two modulated light sources. In addition, for theembodiments of the present invention where three light sources S1, S2,S3 having different light wavelengths are used, there are importantadvantages having one light source S1 not modulated. The advantagesdescribed above for two light sources where one light source is notmodulated can be applied to three light sources embodiments described inthis paragraph.

In still another embodiment of the invention, four excitation lightsources having different wavelength can be used. In still anotherembodiment of the invention, five excitation light sources havingdifferent wavelengths can be used. During operation, at least one lightsource is not modulated and at least one light source isintensity-modulated. For example, in one embodiment, two light sourcesare intensity-modulated and the other two light sources are notmodulated. For such an embodiment, the light beams from the four lightsources may be arranged along the flow cell in such an order that theun-modulated light beam and modulated light team are positioned in analternative manner. The modulation frequencies of twointensity-modulated light sources may be different. In certain preferredembodiment, the modulation frequencies of two intensity-modulated lightsources are the same. For such preferred embodiment of using the samemodulation frequency for two intensity-modulated light sources, theadvantages and benefits described for using a same modulation frequencyfor two modulated light sources in above paragraph for three lightsource embodiments are also applicable here. In addition, for theembodiments where four light sources having different light wavelengthsare used, there are important advantages having one light source notmodulated. The advantages described above for two light sources whereone light source is not modulated can be applied to four light sourcesembodiments described in this paragraph.

In the present invention, at least one of the multiple light sources canbe modulated and at least one of the multiple light sources is notmodulated. For N light sources (N is the number of multiple lightsources with different wavelength), some light sources can be modulatedand other light sources are not-modulated. Preferably, light beams fromthese light sources may be arranged along the flow cell in such an orderthat the un-modulated light beam and modulated light team are positionedin an alternative manner. The modulation frequencies of thoseintensity-modulated light sources may be different. In certain preferredembodiment, the modulation frequencies of the intensity-modulated lightsources are the same. For such N-light source cases, the advantages andbenefits described in previous paragraphs associated with having onelight source not-modulated or with having same modulation frequenciesfor two modulated light sources are also applicable here.

Different methods can be used to modulate light sources, includingdigital modulation and analogue modulation. For digital modulation,light from the light source can be turned on or off by the digitalsignals used for digital modulation with certain modulation ratio (theratio of ON-light intensity to OFF-light intensity). Preferably, thedigital modulation ratio is larger than 10. More preferably, the digitalmodulation ratio is larger than 100. For many practical light sources(e.g. lasers), digital modulation could not achieve a true OFF of lightsource. Thus, digital modulation is a special case of light-intensitymodulation. In analogue modulation, light intensity from the lightsource can be controlled to be proportional to the amplitude of theanalogue signals used for analogue modulation. The analogue signals usedfor modulation can be of different waveforms, including sine-waveform,triangular-waveform, and seesaw-waveform. The light source can bemodulated at any suitable frequencies, as long as the modulatedparticle-induced electronic pulses can be de-modulated throughelectronics-processing means to recover the particle-induced electronicpulses.

The modulation frequency could be in various ranges, typically MHz, fromless than 0.1 MHz to 10 MHz, even above 100 MHz. The modulationfrequency depends on a number of factors, including how fast theparticles/cells are flowing through the flow channel, up to whatfrequency ranges the light source could be possibly modulated and whatde-modulation means is used to recover particle-induced electronicpulses. For example, if the particle/cell flow rate in the flow channelis such that it takes about one to several micro-seconds for aparticle/cell to go through one OIZ, then the modulation frequency couldbe in the range of several MHz or above. On the other hand, if theparticle/cell flow rate is slow and it takes about 10 micro-seconds ormore for a particle/cell to go through one OIZ, then the modulationfrequency could be in the range of 1 MHz or above. High modulationfrequency could be used as long as it is within modulation frequencyrange of the light source and as long as the modulated signals could bede-modulated through electronics-processing means. Preferably, themodulation frequency is between 0.1 MHz and 100 MHz. More preferably,the modulation frequency is between 1 MHz and 20 MHz. Even morepreferably, the modulation frequency is between 2 MHz and 10 MHz. Evenmore preferably, the modulation frequency is between 3 MHz and 8 MHz.

The light illumination sub-system is constructed so that multiple lightbeams of different wavelengths having appropriate geometrical shapes(e.g. elliptical shape, rectangular shape) are directed to multiple OIZsat different locations along the flow direction of the flow channel.That is to say, adjacent optical interrogation zones (or adjacent lightbeams of different wavelengths) do not overlap in the flow channel. Todefine the separation between adjacent optical interrogation zones, thecenter-to-center distance between adjacent optical interrogation zonesis used. Such a center-to-center distance between adjacent OIZs could beof different values. The center-to-center distance could be as large asmore than 500 microns. The center-to-center distance between adjacentlight beams depends on a number of factors including the minor axis oflight beams along the flow direction at OIZs, the light collectionefficiency for light emitted from different OIZs, the flow speed rangeof particles/cells flowing inside the flow channel and the particle/cellconcentration range. For the present invention, singlelight-collection-optics set is used to collect light from multiple OIZsat different locations along the flow channel, light collectionefficiency may differ for light emitted from different OIZs For example,light-collection-optics may include an objective lens or lens set. Thelight collection efficiency may be low for collecting light from OIZsfar away from the central axis of the objective lens or lens set. Thus,the center-to-center distance between adjacent OIZs should not be toolarge so that light collection efficiency for light emitted from anyOIZs is not too low. In addition, a large center-to-center distancebetween adjacent OIZs increases the probability of two particles/cellspassing through two adjacent OIZs simultaneously, especially whenparticle/cell concentration is high. Thus, preferably, at any givenmoment, there should be one or zero particle/cell passing through eitherone of two adjacent optical interrogation zones in the presentinvention. On the other hand, preferably, the light beams at adjacentOIZs do not overlap. For example, if the excitation beam has anelliptical shape whose minor axis is 15 microns parallel to the flowdirection, the center-to-center distance between adjacent OIZs ispreferably between 15 microns and 150 microns, leaving the gap betweenthe adjacent light beams propagating through the flow cells in the rangeof 0 to 135 microns. In one embodiment, if the center-to-center distanceis 30 microns for such 15 micron minor axis beams, then the gap betweenthe adjacent light beams is 15 microns.

In one preferred embodiment, light beams illuminated to the OIZs have aminor axis of about 10 microns. In such an embodiment, preferably, thecenter-to-center distance between adjacent optical interrogation zonesalong the flow direction is between 10 micros and 300 microns. Even morepreferably, the center-to-center distance between adjacent opticalinterrogation zones along the flow direction is between 20 microns and200 microns. Still more preferably, the center-to-center distancebetween adjacent optical interrogation zones along the flow direction isbetween 25 microns and 100 microns. Still more preferably, thecenter-to-center distance between adjacent optical interrogation zonesalong the flow direction is between 30 microns and 80 microns. Stillmore preferably, the center-to-center distance between adjacent opticalinterrogation zones along the flow direction is between 35 microns and70 microns. Note that for light beams having 10 micron as minor axisalong the flow direction, the center-to-center distance of 20 micronsmean that there is no gap between the two adjacentoptical-detection-zones and the distance of 50 microns means that thegap between the two adjacent optical-detection-zones is 30 microns. Thelight illumination sub-system of the present invention may includebeam-steering and/or beam combining and/or light focusing and/or orother optical components to deliver light beams to different locationsalong the flow channel.

Light source system may include components capable of generating lightsuch as laser, light-emitting diodes (LED) or other sources. It may alsoinclude light beam shaping and beam steering optical components. Thosewho are skilled in the art of light beam shaping can readily designdifferent beam shaping optic components and sub-system to shape thelight from the source to desired shape, typically an elliptical ornearly-rectangular shape. Various methods could be used for steeringlight or directing light to multiple optical interrogation zones atdifferent locations along the flow channel.

Particles/cells to be detected or measured are suspended in a liquid andparticle/cell suspension would be flown through the flow channel anddriven into the central region of the flow channel under hydrodynamicfocusing or other focusing forces. When the focused particles/cells passthrough each optical interrogation zone, fluorescent molecules withinthe particles/cells are excited by light beams in the OIZ and producetransient fluorescent light pulses. Similarly, there are alsoparticle-scatter-light-induced pulses as particles/cells go through anOIZ. At the detection side of each channel, the particle-inducedelectronic signal correlated with particle fluorescent intensity andparticle scatter light intensity is generated using a photodetector suchas PMT, APD or the like.

Fluorescent light emitted from multiple optical interrogation zones(OIZ1 and OIZ2 FIG. 3) can be collected via various optical components.Those who are skilled in optical design of flow cytometer and/or inoptical design for light collection could readily design/develop asuitable optical collection sub-system for efficient collection oflight. Among other components, such optical collection sub-system mayinclude optical components such as light-splitting components based onlight wavelengths, such as dichroic mirrors, band-pass filters, low-passfilters and/or high-pass filters, photodetectors such as PMT, APD or thelike. A critical difference from the first approach in prior art (asillustrated in FIG. 1) is that in present invention, there is no needfor optical collection sub-system to physically/optically separate lightfrom different optical interrogation zones to different locations. Thatis, optical collection components can be shared between OIZs due to theintensity modulation approach of the invention. Collected light is thensplit or filtered so that light of various wavelength ranges isseparated and detected by photodetectors such as PMT or APD or the like.Furthermore, for the present invention, light splitting optics includingcomponents such as dichroic mirrors and band-pass filters can also beshared for detecting light emitted from different OIZs.

The electronic output from photodetectors is then processedelectronically. The electronic signals from each channel (i.e. a fixedwavelength range signal) may have multiple components since the light isemitted from multiple OIZs at different locations along the flowchannel. For example, the electronic signals will contain two componentssince light emitted from two OIZs are mixed together and collectedtogether using the same light-collection optics. If different types offluorescent molecules being excited at different OIZs by light sourcesof different wavelengths do not have the same emission spectra (i.e.detection wavelength ranges for theses FMs do not overlap), theisolation of electronic pulses for different fluorescent molecules ispossible since light of different wavelengths is detected by differentphotodetectors (i.e. different detection channel). On the other hand, ifdifferent types of fluorescent molecules being excited at different OIZsby light sources of different wavelengths have the same emission spectra(i.e. detection wavelength ranges for these FMs are the overlapped), theisolation of electronic pulses for different fluorescent molecules isstill possible through de-modulation of the modulated electronic signalsfrom photodetectors. The isolation of electronic pulses for differentfluorescent molecules excited at different OIZs by light sources ofdifferent wavelengths may also be achieved through exploiting the timedifferences of these electronic pulse profiles. Such time differencesare dependent on spatial distances between two OIZs of interest, and onthe travel speed of particle/cell in the flow cell. Therefore, the sameset of filters and photodetector could be shared for detection ofemitted light with the same/overlapped emission spectra but excited bydifferent light sources.

Example 1: Two Intensity-Modulated Light Sources

Below we will discuss some examples of modulation and de-modulationmethods. For example, light beam directed to OIZ1 is modulated by asine-wave signal at a frequency f₁ and phase value zero (i.e. Mod1=sin(2πf₁t)+1) and light beam directed to OIZ2 is modulated by asine-wave signal at a frequency f₂ and a phase value φ₂ (i.e. Mod2=sin(2πf₂t+φ₂)+1)). The first modulation signal Mod 1 has a phase valueof zero and its phase is used as a reference for all signals in thesystem. Thus, the second modulation signal Mod 2 has a phase value ofφ₂. Total electronic signals Total_Signal from a photodetector is thesum of the signal associated with emitted light from OIZ1 (Sig1) andOIZ2 (Sig2), respectively, and can be expressed as,Total_(signal)=Sig1+Sig2=S ₁(t)(sin(2πf ₁ t+φ ₃)+1)+S ₂(t)(sin(2πf ₂ t+φ₄)+1)  (1)where S₁(t) and S₂(t) are output electronic signal from thephotodetector due to particles/cells passing through the 1^(st) OIZ andthe 2^(nd) OIZ, respectively, when no modulation is applied to eitherone of the light sources. Note that in equation (1), the electronicsignals from the photodetector have phase values φ₃ and φ₄, incomparison with phase values of zero and φ₂ of the modulation signals.The factors contributing to such a phase change (or phase difference)include the response-time-delay of light source between the modulationelectronic signals and the modulated light beams at OIZs, the responsetime or relaxation time or life time of fluorescent molecules, and theresponse time of photodetectors, and other possible time delays withinthe system from modulation of light sources to detecting fluorescentsignals on the photodetectors. U.S. Pat. Nos. 5,196,709 and 5,270,548described the method and apparatus capable of measuring the life time offluorescent molecules; the methods of which are herein incorporated byreference. For the present invention, an electronic processor is used tode-modulate the Total_Signal to recover electronic signal S₁(t) andS₂(t) so that the light intensity (i.e. fluorescence or side-scatter)can be derived at the corresponding detection channel.

Different methods may be used for the de-modulation. Below, we willconsider two approaches. In the first approach as illustrated later, wedo not take into account the phase differences between the modulationsignals applied to excitation light beams and the electronic signalsfrom the photodetector. In another word, it is assumed that there is nophase change or phase difference for the electronic signals from thephotodetector, relative to the phase values of the modulation signals.Such a first approach is called “one-component de-modulation”. In thesecond approach, we consider the phase differences between themodulation signals applied to excitation light beams and the electronicsignals from the photodetector. Such a second approach is called“quadrature modulation”.

One-Component De-Modulation

For the first approach of “one-component de-modulation” where the phasedifference between the electronic signals from photodetector and themodulation signals is not considered, total electronic signalsTotal_Signal from a photodetector in equation (1) can be expressed as

$\begin{matrix}\begin{matrix}{{Total}_{Signal} = {{{Sig}\; 1} + {{Sig}\; 2}}} \\{= {{{S_{1}(t)}( {{\sin( {2\pi\; f_{1}t} )} + 1} )} + {{S_{2}(t)}{( {{\sin( {{2\pi\; f_{2}t} + \varphi_{2}} )} + 1} ).}}}}\end{matrix} & (2)\end{matrix}$

In order to recover S₁(t), we would multiply Total_Signal with itscorresponding modulation signal sin(2πf₁t), then we have

$\begin{matrix}\begin{matrix}{{Intermediate\_ Signal} = {{Total\_ Signal}*{\sin( {2\pi\; f_{1}t} )}}} \\{= {{{S_{1}(t)}{\sin( {2\pi\; f_{1}t} )}*{\sin( {2\pi\; f_{1}t} )}} + {{S_{1}(t)}{\sin( {2\pi\; f_{1}t} )}} +}} \\{{{S_{2}(t)}{\sin( {{2\pi\; f_{2}t} + \varphi_{2}} )}*{\sin( {2\pi\; f_{1}t} )}} +} \\{{S_{2}(t)}{\sin( {2\pi\; f_{1}t} )}} \\{= {{0.5{S_{1}(t)}} - {0.5{S_{1}(t)}{\cos( {4\pi\; f_{1}t} )}} + {{S_{1}(t)}{\sin( {2\pi\; f_{1}t} )}} +}} \\{{0.5{S_{2}(t)}{\cos( {{2\pi\;( {{f_{2}t} - {f_{1}t}} )} + \varphi_{2}} )}} -} \\{{0.5{S_{2}(t)}{\cos( {{2{\pi( {{f_{2}t} + {f_{1}t}} )}} + \varphi_{2}} )}} + {{S_{2}(t)}{\sin( {2\pi\; f_{1}t} )}}}\end{matrix} & (3)\end{matrix}$Thus, the above Intermediate_Signal has multiple components, including aterm S₁(t), and other terms with S₁(t) and S₂(t) being modulated withsine-wave or cosine-wave forms at different frequencies of f₁, 2f₁,(f₁+f₂) or |f₁−f₂|. In order to recover S₁(t) from Intermediate_Signal,we analyze the signal in terms of its frequency spectra. Depending onthe linear velocity of a particle/cell flowing through the flow channel(typically, <10 m/sec) as well as the beam size (10˜20 microns), thetime taken for a particle to pass through the optical detection zonecould vary from ˜1 micro-second to more than 1 micron-second, leading tothe frequency bandwidth of the particle-induced electronic pulse signalS₁(t) or S₂(t) being estimated as between 0 and 1 MHz (or even between 0and 0.5 MHz). Thus, the six component terms in the Intermediate_Signalof equation (2) have signal bandwidths between 0 and 1 MHz, between 2f₁and (2f₁+1) MHz, between f₁ and (f₁+1) MHz, between |f₁−f₂| and|f₁−f₂|+1 MHz, between (f₂+f₁) and (f₂+f₁)+1 MHz, and between f₁ and(f₁+1) MHz, respectively. Under these conditions, the modulationfrequency is preferably to be higher than 2-times of the signalfrequency bandwidth, i.e., above 2 MHz. In addition, the differencebetween two modulation frequencies is also preferably to be higher than2-time of the signal frequency bandwidth. In one example, f₁ and f₂could be 6 MHz and 3 MHz, respectively. In another example, f₁ and f₂could be 10 MHz and 4 MHz, respectively. Under these conditions,particle-induced electronic pulse signal S₁(t) could be recovered byfiltering the Intermediate_Signal through a low-pass filter with acut-off frequency at ˜1 MHz. Therefore, except the 1^(st) term, all theother terms in equation (2) would be filtered out by the low-passfilter, thus recover signal S₁(t) from the Intermediate_Signal.Generally speaking, in order to recover signal S₁(t) from theIntermediate_Signal in equation (3), the modulation frequencies of f₁and f₂ must be chosen so that the values of f₁, 2f₁, (f₁+f₂) and |f₁−f₂|should be all above the frequency bandwidth of the signal S₁(t).Preferably, the modulation frequencies of f₁ and f₂ must be chosen sothat the values of f₁ and |f₁−f₂| are at least two-times of thefrequency bandwidth of the signal S₁(t).

Similarly, using such a “one-component de-modulation method”, one couldde-modulate Total_Signal to recover the particle induced electronicpulse signal S₂ (t). FIGS. 4A-4H show the process of the analogmodulation and de-modulation here. FIGS. 4A and 4B show the analogsignals used to modulate the 1^(st) and 2^(nd) light source,respectively. In this example, modulation frequency for the 1^(st) and2^(nd) light source is 3 MHz (f₁) and 6 MHz (f₂), respectively. FIG. 4Cshows the total detected electronic pulse signal if no modulation isemployed for either light source. A particle/cell passes through the1^(st) OIZ and 2^(nd) OIZ sequentially and generate the 1^(st) and2^(nd) pulse, respectively. In this example, we assume thatparticle/cell diameter is 10 microns, passing through the OIZs with a 15micron along the particle/cell flowing direction, and the linearvelocity of the particle/cell is assumed to be 5 m/sec. Furthermore, weassume that the center-to-center distance between these two OIZs is 30microns. Therefore, the width of the generated individualparticle-induced electronic pulse is estimated to be 5 μs (i.e. (10μm+15 μm)/(5 m/s)) and the separation between two pulse peaks isestimated to be 6 μs (i.e. 30 μm/(5 m/s). FIG. 4D shows the totaldetected electronic signals when both light sources are modulated usingthe modulation signals of FIG. 4A and FIG. 4B, respectively. FIGS. 4Eand 4F illustrates the intermediate signal when de-modulation is appliedto the total signal of FIG. 4D to recover the 1^(st) pulse profile and2^(nd) pulse profile, respectively. FIGS. 4G and 4H show the recovered1^(st) and 2^(nd) electronic pulse profiles, respectively, generated asthe particle passes through the 1^(st) and 2^(nd) light beams at twoOIZs. Note that an amplification/scaling factor of 2 is applied to thesignals on FIGS. 4G and 4H after the signals at FIGS. 4E and 4F arefiltered by a low-pass filter with a cut-off frequency of 1 MHz.

Quadrature De-Modulation

In a second approach of quadrature demodulation, we consider the phasedifferences between the modulation signals applied to excitation lightbeams and the electronic signals from the photodetector. The totalelectronic signals Total_Signal from a photodetector in equation (1) isas follows:Total_(signal) =S ₁(t)(sin(2πf ₁ t+φ ₃)+1)+S ₂(t)(sin(2πf ₂ t+φ₄)+1).  (4)In order to recover S₁(t), we would multiply Total_Signal with itscorresponding modulation signal sin(2πf₁t) and a 90-degree phase-shiftsignal cos(2πf₁t), then we have obtained two Intermediate_Signal_1 andIntermediate_Signal_2

$\begin{matrix}\begin{matrix}{{{Intermediate\_ Signal}\_ 1} = {{Total\_ Signal}*{\sin( {2\pi\; f_{1}t} )}}} \\{= {{{S_{1}(t)}{\sin( {{2\pi\; f_{1}t} + \varphi_{3}} )}*{\sin( {2\pi\; f_{1}t} )}} +}} \\{{{S_{1}(t)}{\sin( {2\pi\; f_{1}t} )}} + {{S_{2}(t)}{\sin( {{2\pi\; f_{2}t} + \varphi_{4}} )}*}} \\{{\sin( {2\pi\; f_{1}t} )} + {{S_{2}(t)}{\sin( {2\pi\; f_{1}t} )}}} \\{= {{0.5{S_{1}(t)}{\cos( \varphi_{3} )}} - {0.5{S_{1}(t)}{\cos( {{4\pi\; f_{1}t} + \varphi_{3}} )}} +}} \\{{{S_{1}(t)}{\sin( {2\pi\; f_{1}t} )}} +} \\{{0.5{S_{2}(t)}{\cos( {{2\pi\;( {{f_{2}t} - {f_{1}t}} )} + \varphi_{4}} )}} -} \\{{0.5{S_{2}(t)}{\cos( {{2{\pi( {{f_{2}t} + {f_{1}t}} )}} + \varphi_{4}} )}} +} \\{{S_{2}(t)}{\sin( {2\pi\; f_{1}t} )}}\end{matrix} & (5) \\\begin{matrix}{{{Intermediate\_ Signal}\_ 2} = {{Total\_ Signal}*{\cos( {2\pi\; f_{1}t} )}}} \\{= {{{S_{1}(t)}{\sin( {{2\pi\; f_{1}t} + \varphi_{3}} )}*{\cos( {2\pi\; f_{1}t} )}} +}} \\{{{S_{1}(t)}{\cos( {2\pi\; f_{1}t} )}} + {{S_{2}(t)}{\sin( {{2\pi\; f_{2}t} + \varphi_{4}} )}*}} \\{{\cos( {2\pi\; f_{1}t} )} + {{S_{2}(t)}{\cos( {2\pi\; f_{1}t} )}}} \\{= {{0.5{S_{1}(t)}{\sin( \varphi_{3} )}} + {0.5{S_{1}(t)}{\sin( {{4\pi\; f_{1}t} + \varphi_{3}} )}} +}} \\{{{S_{1}(t)}{\cos( {2\pi\; f_{1}t} )}} +} \\{{0.5{S_{2}(t)}{\sin( {{2\pi\;( {{f_{2}t} + {f_{1}t}} )} + \varphi_{4}} )}} + {0.5{S_{2}(t)}}} \\{{\sin( {{2{\pi( {{f_{2}t} - {f_{1}t}} )}} + \varphi_{4}} )} + {{S_{2}(t)}{\cos( {2\pi\; f_{1}t} )}}}\end{matrix} & (6)\end{matrix}$

Thus, the above Intermediate_Signal_1 in equation (5) has multiplecomponents, including a term S₁(t)cos(φ₃), and other terms with S₁(t)and S₂(t) being modulated with sine-wave or cosine-wave forms atdifferent frequencies of f₁, 2f₁, (f₁+f₂) or |f₁−f₂|. Similarly,Intermediate_Signal_2 in equation (6) has multiple components, includinga term S₁(t)sin(φ₃), and other terms with S₁(t) and S₂ (t) beingmodulated with sine-wave or cosine-wave forms at different frequenciesof f₁, 2f₁, (f₁+f₂) or |f₁−f₂|. Similar to Intermediate_Signal inequation (3), by choosing appropriate values of modulation frequenciesof f₁ and f₂ so that the values of f₁ and |f₁−f₂| are preferably atleast two-times of the frequency bandwidth of the signal S₁(t),Intermediate_Signal_1 in equation (5) and Intermediate_Signal_2 inequation (6) can be processed through a low-pass filter to obtain termsof term S₁(t)cos(φ₃) and term S₁(t)sin(φ₃), respectively. Thus, signalS₁(t) can be calculated throughS ₁(t)=√{square root over ((S ₁(t)cos(φ₃))²+(S ₁(t)sin(φ₃))²)}  (7)

Above procedure in the so-called quadrature de-modulation involves themultiplications of total electronic signals with both modulation signalsin(2πf₁t) and a 90-degree phase-shift signal cos(2πf₁t), it is capableof de-modulation of phase-shifted electronic signal components.

Similarly, using such a “quadrature de-modulation method”, one couldde-modulate Total_Signal in equation (5) to recover the particle inducedelectronic pulse signal S₂ (t).

The single-component de-modulation and quadrature de-modulation methodsdescribed above were for the cases of two modulated light beams directedtwo OIZs in a flow cell. Such methods could be readily extended to threeor more modulated light beams focused onto corresponding numbers of OIZsin a flow cell.

Furthermore, whilst above description of single-component de-modulationand quadrature de-modulation was based on modulation of light beamsusing a sine-wave, these de-modulation methods are also applicable toother waveform based modulations. According to Fourier transformprinciple, all periodic waveforms could be decomposed into the summationof multiple sine-wave forms having DC component (constant component),1^(st) harmonics, 2^(nd) harmonics, 3^(rd) harmonics etc. Thus, totalsignal in above equation (2) and (4) would include more sine-wave termsat all these harmonic frequencies, when the light source is modulatedwith waveforms other than sine-waves. During de-modulation processes,similar multiplication step (as shown in equations (3), (5) and (6))would be taken where total signal in equation (2) and (4) is multipliedby sine or cosine functions at 1^(st) harmonic frequency (assuming thatthe 1^(st) harmonic component magnitude is to be recovered since formany periodic waveforms, 1^(st) harmonic would have the largestamplitude among all harmonics). Using similar low-pass filter, one canrecover the electronic pulse profile caused by particle/cell passingthrough an optical interrogation zone. It is worthwhile to describe thatthe selection of appropriate modulation frequencies is especiallyimportant when modulation signals are taken non-sine waveforms, in orderto effectively recover signals through low-pass filtering of theintermediate signals during de-modulation process. As described abovefor intermediate signals of equations (3), (5) and (6) when modulationsignals are of sine waveforms, the modulation frequencies of f₁ and f₂should be chosen properly so that the values of f₁ and (f₁−f₂) arepreferably at least two-times of the frequency bandwidth of the signalS₁(t). When a non-sine waveform is used for modulation, for example, asquare waveform or a digital modulation may be applied to modulate lightsources due to the possibility of higher-frequency modulation. Assumethat two modulation waveforms have the first harmonic frequencies off_(h1) and f_(h2). The intermediate signals after multiplying the totaldetected signals with the modulation 1^(st) harmonic signals wouldresult in a signal term and other signals terms being modulated withsine-wave or cosine-wave forms at different frequencies of f_(h1),2f_(h1), 3f_(h1), . . . nf_(h1), . . . , and (f_(h1)+f_(h2)),(f_(h1)+2f_(h2)), . . . (f_(h1)+nf_(h2)), . . . and |f_(h1)−f_(h2)|,|f_(h1)−2f_(h2)|, . . . |f_(h1)−nf_(h2)|, . . . etc. Thus when two lightsources are modulated at two different frequencies (harmonics beingf_(h1) and f_(h2)), the modulation frequencies of f_(h1) and f_(h2)should be chosen properly values of f₁ and |f_(h1)−f_(h2)|,|f_(h1)−2f_(h2)|, . . . |f_(h1)−nf_(h2)|, . . . , are all preferably atleast two-times of the frequency bandwidth of the signal S₁(t). This isan important requirement for effective recovery and filtering of thesignal S₁(t) using the de-modulation methods described above.

In the present invention, light beams from different light sources arefocused onto or directed to different optical interrogation zones(OIZs). Thus, for above examples, electronic signal component S₁(t) andS₂(t) would occupy different time windows, as long as there aresufficient distances between two OIZs and as long as the gap between twoOIZs is large than particle diameter. Such a spatial separation betweendifferent OIZs and time-domain separation between signal componentsS₁(t) and S₂(t) are preferred, and have special advantages and benefitscompared with the condition where light beams from different OIZs areoverlapping or coincide with each other. Firstly, if two (or more) lightbeams from different light sources are directed to the same OIZ,different fluorescent molecules that can be excited by differentwavelengths would be excited simultaneously as particle/cell passesthrough the OIZ. If their fluorescent emission spectra overlap, thenfluorescent signals from different fluorescent molecules would bedetected by the same photodetector. Thus, the dynamic range of thephotodetector would be shared by the measurements on two or morefluorescent molecules. As a result, each fluorescent molecule may occupyonly partial ranges of the complete dynamic range. On the other hand,for the present invention, light beams from different light sources arefocused onto or directed to different optical interrogation zones(OIZs). Thus, no two fluorescent molecules would be excitedsimultaneously if they are excitable by two different light wavelengths.In another word, even if these two or more fluorescent molecules emitthe same emission spectra range and are detected by the samephotodetector, each type of fluorescent molecule is still able toutilize entire dynamic range of the photodetector. Secondly, when lightbeams from different light sources coincide and are directed onto thesame OIZ, even if these light beams are modulated at differentfrequencies, signals for different fluorescent molecules may interferewith each other. As a result, there will be cross-talks betweenelectronic signals corresponding to different fluorescent molecules,even if these fluorescent molecules are excited by different lightsources. Consider the following example where light beam from 1^(st)light source at one light wavelength is modulated at frequency f₁ andlight beam from 2^(nd) light source at a different wavelength ismodulated at frequency f₂ and both light beams are directed to the sameoptical interrogation zone (OIZ). When a particle containing one type offluorescent molecule to be excited by the 1^(st) light source passesthrough the OIZ, theoretically there should be only electronic signalsat frequency f₁, corresponding to excitation of this type of fluorescentmolecules in the particle/cell. In practice, due to the noises in the1^(st) light source, in the photodetector and in associated electroniccircuits, there will be noise components occurring at frequency f₂,which will be detected as “signal from some other fluorescent moleculesbeing excited by the 2^(nd) light source”. Importantly, suchnoise-signal amplitudes at frequency f₂ may increase with the realsignal amplitude associated with the fluorescent molecules in theparticle/cell, due to noise natures in the photodetector and associatedcircuits. For example, it is well-known that a photodetector would havelarger noises in amplitudes when it is exposed to a larger-intensitylight signal. On the other hand, when light beams from different lightsources are directed to different optical interrogation zones (OIZs),there will be no such direct interference of signals for differentfluorescent molecules excited by different light beams with differentwavelengths at different locations. Electronic signals having modulationfrequency f₁ would be detected from photodetectors only for the timewindow when the particle travels through OIZ1 and will not be detectedfor other time ranges when the particle travels outside OIZ1. Similarly,electronic signals having modulation frequency f₂ would be detected fromphotodetectors only for the time window when particle travels throughOIZ2 and will not be detected for other time ranges when the particle isoutside OIZ2. For the particle that contains only fluorescent moleculesbeing excited by the 1^(st) light beam in OIZ1, there will be no signalassociated with 2^(nd) light excitation source when it travels throughOIZ1, even though the noises would be larger during such a time windowsince particle's fluorescent molecules are excited by the 1^(st) lightbeam and would emit lights to be detected at the photodetector. Whenthis particle moves through OIZ2, there would be minimum or no signal atall corresponding to the 2^(nd) light beam since it does not containfluorescent molecules being excited by the 2^(nd) light source. Theminimum signal, if any, at such time window, is simply associated withdark current of photodetector and other background noises of thecircuits and other system components.

Example 2: One Intensity-Modulated Light Source and One Non-ModulatedLight Source

In above sections, both light beams are modulated and single-componentde-modulation and quadrature demodulation methods are used to recovercorresponding electronic pulse profiles from the modulated signals. Infollowing examples, we consider the cases where one light beam is notmodulated and modulation is applied to the other light beams. We performexemplary analysis for the case of two excitation sources where lightbeam directed to OIZ1 is modulated by a sine-wave signal at a frequencyf₁ and phase value zero (Mod 1=sin(2πf₁t)+1) and light beam directed toOIZ2 is not modulated. Total electronic signals Total_Signal from aphotodetector is the sum of the signal associated with emitted lightfrom OIZ1 (Sig1) and OIZ2 (Sig2), respectively, and can be expressed as,Total_Signal=Sig1+Sig2=S ₁(t)(sin(2πf ₁ t+φ ₃)+1)+S ₂(t)  (8)where S₁(t) and S₂(t) are output electronic signal from thephotodetector due to particles/cells passing through the 1^(st) OIZ andthe 2^(nd) OIZ, respectively, when no modulation is applied to eitherone of the light sources. An electronic processor is used to de-modulatethe Total_Signal to recover electronic signal S₁(t) and S₂(t) so thatthe light intensity (i.e. fluorescence or side-scatter) can be derivedat the corresponding detection channel. Similar to the example of twomodulated light sources, both single-component de-modulation method andquadrature de-modulation method can be used here to recover electronicsignals signal S₁(t) and S₂(t).Single-Component De-Modulation

For single component de-modulation, we would multiply Total_Signal withits corresponding modulation signal sin(2πf₁t), then we have

$\begin{matrix}\begin{matrix}{{Intermediate\_ Signal} = {{Toatl\_ Signal}*{\sin( {2\pi\; f_{1}t} )}}} \\{= {{{S_{1}(t)}{\sin( {{2\pi\; f_{1}t} + \varphi_{3}} )}*{\sin( {2\pi\; f_{1}t} )}} +}} \\{{{S_{1}(t)}{\sin( {2\pi\; f_{1}t} )}} + {{S_{2}(t)}{\sin( {2\pi\; f_{1}t} )}}} \\{= {{0.5{S_{1}(t)}\cos\;\varphi_{3}} - {0.5{S_{1}(t)}{\cos( {{4\pi\; f_{1}t} + \varphi_{3}} )}} +}} \\{{{S_{1}(t)}{\sin( {2\pi\; f_{1}t} )}} + {{S_{2}(t)}{\sin( {2\pi\; f_{1}t} )}}}\end{matrix} & (9)\end{matrix}$

Thus, the above Intermediate_Signal has multiple components, including aterm S₁(t), and other terms with SAO and S₂(t) being modulated withsine-wave or cosine-wave forms at different frequencies of f₁, or 2f₁.Similar to the analysis described above for equation (3), the modulationfrequency f₁ should be chosen to be at least two times of signalbandwidth. For example, the signal bandwidth may be about 1 MHz and f₁could have a value of 3 MHz. Using a low pass filter with acut-frequency of, e.g. 1 MHz, particle-induced electronic pulse signalS₁(t) could be recovered by filtering the Intermediate_Signal.Therefore, except the 1^(st) term, all the other terms in equation (9)would be filtered out by the low-pass filter, thus recover signal S₁(t)cos φ₃ from the Intermediate_Signal. Note that phase angle φ₃ reflectsthe phase difference of the electronic signal component at modulationfrequency f₁ from the photodetector, relative to the phase of modulationsignal itself (phased at zero as reference). As described above, thefactors contributing to such a phase change (or phase difference)include the response-time-delay of light source between the modulationelectronic signal and the modulated light beam at the corresponding OIZ,the response time or relaxation time or life time of fluorescentmolecules, and the response time of photodetectors, and other possibletime delays within the system from modulation of light sources todetecting fluorescent signals on the phtodetectors. U.S. Pat. Nos.5,196,709 and 5,270,548 described the method and apparatus capable ofmeasuring the life time of fluorescent molecules; the disclosure ofwhich is herein incorporated by reference.

In this approach of “one-component de-modulation”, we do not take intoaccount the phase differences between the modulation signals applied toexcitation light beams and the electronic signals from thephotodetector. In another word, it is assumed that there is no phasechange or phase difference for the electronic signals from thephotodetector, relative to the phase values of the modulation signals.Under such a consideration, phase angle φ₃ can be approximated as zero.Thus, particle induced electronic pulse signal S₁(t) is recovered(S₁(t)cos φ₃=S₁(t)).

Furthermore, particle-induced electronic pulse signal S₂(t) could alsobe recovered by filtering Total_Signal in equation (8) with such a lowpass filter. Therefore, except the 1^(st) term, all the other terms inequation (8) would be filtered out by the low-pass filter, thus recoversignal S₂(t) from the Total_Signal.

FIGS. 5A-F show the process of the analog modulation and de-modulationdescribed in this example. FIG. 5A shows an analog signal used tomodulate the 1^(st) light source. In this example, the modulationfrequency for the 1^(st) light source is 3 MHz (f₁). FIG. 5B shows thetotal detected electronic pulse signal if no modulation is employed forthe 1^(st) light source. A particle/cell passes through the 1^(st) OIZand 2^(nd) OIZ sequentially and generate the 1^(st) and 2^(nd) pulse,respectively. In this example, we assume that particle/cell diameter is10 microns, passing through the OIZs with a 15 micron along theparticle/cell flowing direction, and the linear velocity of theparticle/cell is assumed to be 5 m/sec. Furthermore, we assume that thecenter-to-center distance between these two OIZs is 30 microns.Therefore, the width of the generated individual particle-inducedelectronic pulse is estimated to be 5 μs (i.e. (10 μm+15 μm)/(5 m/s))and the separation between two pulse peaks is estimated to be 6 μs (i.e.30 μm/(5 m/s). FIG. 5c shows the total detected electronic signals whenthe 1^(st) light source is modulated using the modulation signal of FIG.5A. FIG. 5D illustrates the intermediate signal when de-modulation isapplied to the total signal of FIG. 5C to recover the 1^(st) pulseprofile. FIGS. 5E and 5F show the recovered 1^(st) and 2^(nd) electronicpulse profiles, respectively, generated as the particle passes throughthe 1^(st) and 2^(nd) light beams at two OIZs. Note that anamplification/scaling factor of 2 is applied to the signals on FIG. 5Eafter the signal at FIG. 5D is filtered with a low-pass filter with acut-off frequency of 1 MHz.

Quadrature De-Modulation

For quadrature demodulation, we consider the phase differences betweenthe modulation signals applied to excitation light beams and theelectronic signals from the photodetector. In order to recover S₁(t)from equation (8), we would multiply Total_Signal in equation (8) withits corresponding modulation signal sin(2πf₁t) and a 90-degreephase-shift signal cos(2πf₁t), then we have obtained twoIntermediate_Signal_1 and Intermediate_Signal_2

$\begin{matrix}{\mspace{25mu}\begin{matrix}{{{Intermediate\_ Signal}\_ 1} = {{Toatl\_ Signal}*{\sin( {2\pi\; f_{1}t} )}}} \\{= {{{S_{1}(t)}{\sin( {{2\pi\; f_{1}t} + \varphi_{3}} )}*{\sin( {2\pi\; f_{1}t} )}} +}} \\{{{S_{1}(t)}{\sin( {2\pi\; f_{1}t} )}} + {{S_{2}(t)}{\sin( {2\pi\; f_{1}t} )}}} \\{= {{0.5{S_{\; 1}(t)}\cos\;\varphi_{3}} -}} \\{{0.5{S_{1}(t)}{\cos( {{4\pi\; f_{1}t} + \varphi_{3}} )}} +} \\{{{S_{1}(t)}{\sin( {2\pi\; f_{1}t} )}} + {{S_{2}(t)}{\sin( {2\pi\; f_{1}t} )}}}\end{matrix}} & (10) \\\begin{matrix}{{{Intermediate\_ Signal}\_ 2} = {{Toatl\_ Signal}*{\cos( {2\pi\; f_{1}t} )}}} \\{= {{{S_{1}(t)}{\sin( {{2\pi\; f_{1}t} + \varphi_{3}} )}*{\cos( {2\pi\; f_{1}t} )}} +}} \\{{{S_{1}(t)}{\cos( {2\pi\; f_{1}t} )}} + {{S_{2}(t)}{\cos( {2\pi\; f_{1}t} )}}} \\{= {{0.5{S_{1}(t)}\sin\mspace{2mu}\varphi_{3}} +}} \\{{0.5S_{1}{\sin( {{4\pi\; f_{1}t} + \varphi_{3}} )}} + {{S_{1}(t)}{\cos( {2\pi\; f_{1}t} )}} +} \\{{S_{2}(t)}{\cos( {2\pi\; f_{1}t} )}}\end{matrix} & (11)\end{matrix}$

Thus, the above Intermediate_Signal_1 in equation (10) has multiplecomponents, including a term S₁(t)cos(φ₃), and other terms with S₁(t)and S₂(t) being modulated with sine-wave or cosine-wave forms atdifferent frequencies of f₁ or 2f₁. Similarly, Intermediate_Signal_2 inequation (11) has multiple components, including a term S₁(t)sin(φ₃),and other terms with S₁(t) and S₂ (t) being modulated with sine-wave orcosine-wave forms at different frequencies of f₁ or 2f₁. Similar to theanalysis described above for equation (3), the modulation frequency f₁should be chosen to be at least two times of signal bandwidth. Thus,Intermediate_Signal_1 in equation (10) and Intermediate_Signal_2 inequation (11) can be processed through a low-pass filter to obtain termsof term S₁(t) cos(φ₃) and term S₁(t)sin(φ₃), respectively. Thus, signalS₁(t) can be calculated throughS ₁(t)=√{square root over ((S ₁(t)cos(φ₃))²+(S ₁(t)sin(φ₃))²)}  (12)

The single-component de-modulation and quadrature de-modulation methodsdescribed above were for the cases of one modulated light beam and onenon-modulated light beam directed two OIZs in a flow cell. Such methodscould be readily extended to three or more light beams focused ontocorresponding numbers of OIZs in a flow cell, with at least onemodulated beam and one non-modulated beam.

Furthermore, whilst above description of single-component de-modulationand quadrature de-modulation was based on modulation of light beamsusing a sine-wave, these de-modulation methods are also applicable toother waveform based modulations. According to Fourier transformprinciple, all periodic waveforms could be decomposed into the summationof multiple sine-wave forms having DC component (constant component),1^(st) harmonics, 2^(nd) harmonics, 3^(rd) harmonics etc. Thus, totalsignal in above equation (8) would include more sine-wave terms at allthese harmonic frequencies, when the light source is modulated withwaveforms other than sine-waves. During de-modulation processes, similarmultiplication step (as shown in equations (9), (10) and (11)) would betaken where total signal in equation (8) is multiplied by sine or cosinefunctions at 1^(st) harmonic frequency (assuming that the 1^(st)harmonic component magnitude is to be recovered since for many periodicwaveforms, 1^(st) harmonic would have the largest amplitude among allharmonics). Using similar low-pass filter, one can recover theelectronic pulse profile caused by particle/cell passing through anoptical interrogation zone. It is interesting to discuss how appropriatemodulation frequency should be chosen when modulation signals are takennon-sine waveforms, in order to effectively recover signals throughlow-pass filtering of the intermediate signals during de-modulationprocess. As described above for intermediate signals of equations (9),(10) and (11) when modulation signals are of sine waveforms, themodulation frequencies of f₁ should be chosen properly so that the valueof f₁ is preferably at least two-times of the frequency bandwidth of thesignal S₁(t). When a non-sine waveform is used for modulation, forexample, a square waveform or a digital modulation may be applied tomodulate light sources due to the possibility of higher-frequencymodulation. Assume that the modulation waveform has the first harmonicfrequencies of f_(h1). The intermediate signals after multiplying thetotal detected signals with the modulation 1^(st) harmonic signals wouldresult in a signal term and other signals terms being modulated withsine-wave or cosine-wave forms at different frequencies of f_(h1),2f_(h1), 3f_(h1), . . . nf_(h1), etc. Thus, if only one light source ismodulated or if multiple modulated light sources are modulated with thesame modulation signal (thus the same frequency), the modulationfrequencies of f_(h1) should be chosen properly, so that f_(h1) ispreferably at least two-times of the frequency bandwidth of the signalS₁(t).

In the present invention, light beams from different light sources arefocused onto or directed to different optical interrogation zones(OIZs). Thus, for above examples, electronic signal component S₁(t) andS₂(t) would occupy different time windows, as long as there aresufficient distances between two OIZs and as long as the gap between twoOIZs is large than particle diameter. Such a spatial separation betweendifferent OIZs and time-domain separation between signal componentsS₁(t) and S₂(t) are preferred, and have special advantages and benefitscompared with the condition where light beams from different OIZs areoverlapping or coincide with each other. Firstly, if two (or more) lightbeams from different light sources are directed to the same OIZ,different fluorescent molecules that can be excited by differentwavelengths would be excited simultaneously as particle/cell passesthrough the OIZ. If their fluorescent emission spectra overlap, thenfluorescent signals from different fluorescent molecules would bedetected by the same photodetector. Thus, the dynamic range of thephotodetector would be shared by the measurements on two or morefluorescent molecules. As a result, each fluorescent molecule may occupyonly partial ranges of the complete dynamic range. On the other hand,for the present invention, light beams from different light sources arefocused onto or directed to different optical interrogation zones(OIZs). Thus, no two fluorescent molecules would be excitedsimultaneously if they are excitable by two different light wavelengths.In another word, even if these two or more fluorescent molecules emitthe same emission spectra range and are detected by the samephotodetector, each type of fluorescent molecule is still able toutilize entire dynamic range of the photodetector. Secondly, when lightbeams from different light sources coincide and are directed onto thesame OIZ, even if one light beam is modulated and one light is notmodulated, signals for different fluorescent molecules (supposedly dueto different light sources at different wavelengths) may interfere witheach other. Thus, there will be cross-talks between electronic signalscorresponding to different fluorescent molecules. Consider the followingexample where one light beam from 1^(st) light source at one lightwavelength is not modulated and another light beam from 2^(nd) lightsource at a different wavelength is modulated at frequency f₂ and bothlight beams are directed to the same optical interrogation zone (OIZ).When a particle containing one type of fluorescent molecule to beexcited by the 1^(st) light source passes through the OIZ, theoreticallythere should be only electronic signals corresponding to excitation ofthis type of fluorescent molecules in the particle/cell. In practice,due to the noises in the 1^(st) light source, in the photodetector andin associated electronic circuits, there will be electronic noisesoccurring at frequency f₂, which will be detected as “signal from otherfluorescent molecules being excited by the 2^(nd) light source”.Importantly, such noise-signal amplitudes at frequency f₂ may increasewith the real electronic signals corresponding to the excitation of thefluorescent molecules in the particle, due to noise natures in thephotodetector and associated circuits. For example, it is well-knownthat a photodetector would have larger noises in amplitudes when it isexposed to a larger-intensity light signal. On the other hand, whenlight beams from different light sources are directed to differentoptical interrogation zones (OIZs), there will be no such directinterference of signals for different fluorescent molecules excited bydifferent light beams with different wavelengths at different locations.Un-modulated electronic signals would be detected from photodetectorsonly for the time window when the particle travels through OIZ1 and willnot be detected for other time ranges when the particle travels outsideOIZ1. Similarly, electronic signals having modulation frequency f₂ wouldbe detected from photodetectors only for the time window when particletravels through OIZ2 and will not be detected for other time ranges whenthe particle is outside OIZ2. For the particle that contains onlyfluorescent molecules being excited by the 1^(st) light beam in OIZ1,there will be no signal associated with 2^(nd) light excitation sourcewhen it travels through OIZ1, even though the noises would be largerduring such a time window since particle's fluorescent molecules areexcited by the 1^(st) light beam and emit lights onto the photodetector.When this particle moves through OIZ2, there would be minimum or nosignal at all corresponding to the 2^(nd) light beam since it does notcontain fluorescent molecules being excited by the 2^(nd) light source.The minimum signal, if any, at such time window, is simply associatedwith dark current of photodetector and other background noises of thecircuits and other system components.

Example 3: Two Intensity-Modulated Light Sources at Same ModulationFrequency and Different Modulation Phases

In above examples, modulation applied to different light sources hasbeen based on differences in modulation frequency (f₁ and f₂ in equation(1)) and in amplitude (1 for modulation S₁(t) and 0 for S₂ (t) inequation (8)). Modulation of different light sources could also be basedon phase angles of the modulation signals but with the same modulationfrequency. Light beam directed to OIZ1 is modulated by a sine-wavesignal at a frequency f₁ with phase angle 0 (i.e. Mod 1=sin(2πf₁t)+1)and light beam directed to OIZ2 is modulated by a sine-wave signal atthe same frequency f₁ but with a phase angle θ (i.e. Mod2=sin(2πf₁t+0)+1). Total_Signal from a photodetector is the sum of thesignal associated with emitted light from OIZ1 (Sig1) and OIZ2 (Sig2),respectively, and can be expressed as,Total_Signal=Sig1+Sig2=S ₁(t)(sin(2πf ₁ t)+1)+S ₂(t)(sin(2πf ₁t+θ)+1)  (13)where S₁(t) and S₂(t) are output electronic signal from thephotodetector due to particles/cells passing through the 1^(st) OIZ andthe 2^(nd) OIZ, respectively, when no modulation is applied to eitherone of the light sources. Note that for this example where thedifference in modulation signals is based on phase angles, we will notconsider the phase differences between the output electronic signalcomponents and the modulation signals, as being described above inequations (1), (4), and (8). An electronic processor is used tode-modulate the Total_Signal to recover electronic signal S₁(t) andS₂(t) so that the light intensity (i.e. fluorescence or side-scatter)can be derived at the corresponding detection channel. In one exemplaryapproach, two intermediate signals are derived by multiplyingTotal_Signal with a sine-wave signal with the same frequency as themodulation signal (i.e. sin(2πf₁t)) and a cosine-wave signal with thesame frequency as the modulation signal (i.e. cos(2πf₁t)), respectively,then we have

$\begin{matrix}\begin{matrix}{{{Intermediate\_ Signal}\_ 1} = {{Toatl\_ Signal}*{\sin( {2\pi\; f_{1}t} )}}} \\{= {{{S_{1}(t)}{\sin( {2\pi\; f_{1}t} )}*{\sin( {2\pi\; f_{1}t} )}} +}} \\{{{S_{1}(t)}{\sin( {2\pi\; f_{1}t} )}} + {{S_{2}(t)}{\sin( {2\pi\; f_{1}t} )}*}} \\{{{\sin( {2\pi\; f_{1}t} )}*{\cos(\theta)}} + {{S_{2}(t)}{\sin( {2\pi\; f_{1}t} )}*}} \\{{{\cos( {2\pi\; f_{1}t} )}*{\sin(\theta)}} + {{S_{2}(t)}{\sin( {2\pi\; f_{1}t} )}}} \\{= {{0.5{S_{1}(t)}} - {0.5{S_{1}(t)}{\cos( {4\pi\; f_{1}t} )}} +}} \\{{{S_{1}(t)}{\sin( {2\pi\; f_{1}t} )}} + {0.5{S_{2}(t)}{\cos(\theta)}} -} \\{{0.5{S_{2}(t)}{\cos(\theta)}{\cos( {4\pi\; f_{1}t} )}} +} \\{{0.5{S_{2}(t)}{\sin(\theta)}{\sin( {4\pi\; f_{1}t} )}} + {{S_{2}(t)}{\sin( {2\pi\; f_{1}t} )}}}\end{matrix} & (14) \\\begin{matrix}{{{Intermediate\_ Signal}\_ 2} = {{Toatl\_ Signal}*{\cos( {2\pi\; f_{1}t} )}}} \\{= {{{S_{1}(t)}{\sin( {2\pi\; f_{1}t} )}*{\cos( {2\pi\; f_{1}t} )}} +}} \\{{{S_{1}(t)}{\cos( {2\pi\; f_{1}t} )}} + {{S_{2}(t)}{\cos( {2\pi\; f_{1}t} )}*}} \\{{{\sin( {2\pi\; f_{1}t} )}*{\cos(\theta)}} + {{S_{2}(t)}{\cos( {2\pi\; f_{1}t} )}*}} \\{{{\cos( {2\pi\; f_{1}t} )}*{\sin(\theta)}} + {{S_{2}(t)}{\cos( {2\pi\; f_{1}t} )}}} \\{= {{0.5{S_{1}(t)}{\sin( {4\pi\; f_{1}t} )}} + {{S_{1}(t)}{\cos( {2\pi\; f_{1}t} )}} +}} \\{{0.5{S_{2}(t)}{\cos(\theta)}{\sin( {4\pi\; f_{1}t} )}} + {0.5{S_{2}(t)}{\sin(\theta)}} +} \\{{0.5{S_{2}(t)}{\sin( {4\pi\; f_{1}t} )}} + {{S_{2}(t)}{\cos( {2\pi\; f_{1}t} )}}}\end{matrix} & (15)\end{matrix}$

Similar to the analysis described above for equation (3) where themodulation frequency f₁ should be chosen properly to be more than twotimes of the signal bandwidth, low pass filters could be applied tofilter Intermediate_Signal_1 and Intermediate_Signal_2, which results inFiltered_Intermediate_Signal_1=0.5S ₁(t)+0.5 cos(θ)S ₂(t)  (16)andFiltered_Intermediate_Signal_2=0.5 sin(θ)S ₂(t)  (17)Thus, S₁(t) and S₂(t) could be recovered asS ₂(t)=2×Filtered_Intermediate_Signal_2/sin(θ)  (18)S₁(t)=2×Filtered_Intermediate_Signal_1−2×Filtered_Intermediate_Signal_2×cos(θ)/sin(θ)  (19)where phase angle θ should not be zero or 180 degree. Thus, equations(13), (18) and (19) illustrated the method for modulating two lightsources based on phase-angles and the method for de-modulation torecover particle-induced electronic pulse signal S₁(t) and S₂(t).Modulation of different light sources based on different phase anglesbut the same modulation frequency (as described in an example shown inEqn (13) through Eqn (19)) leads to simple electronics for generatingsuch modulation signals, which becomes especially beneficial when thenumber of excitation light sources increases (e.g. ≥3 light sources).Preferably, for using the modulation signals having the same frequencybut different phase angles as discussed in this example, the phase angleθ is preferably selected at 90 degree. Under such preferred embodiments,the low-pass filtered signal components in equations (16) and (17)becomeFiltered_Intermediate_Signal_1=0.5S ₁(t)  (20)andFiltered_Intermediate_Signal_2=0.5S ₂(t)  (21)

Other methods of modulation and de-modulation could also be used formodulating light sources and recovering the modulated electronic pulseprofiles generated as particles/cells pass through multiple opticalinterrogation zones. Those who are skilled in the art of electronics andelectronic signal processing could readily employ various methods ofmodulation and de-modulation to achieve the required functionalitieshere. For example, as mentioned previously, modulation with square-waveforms or triangular-waveforms could also be applied to modulate lightsources.

FIG. 3 illustrates one embodiment of the present invention where theflow cytometry comprises two excitation light sources (S1 and S2). FIG.4 and FIG. 5 illustrate two examples of the method of the presentinvention for detecting the modulated light emitted from two differentOIZs along a flow channel and for de-modulating and processing totaldetected electronic signals to recover and isolate the particle-inducedelectronic pulse profiles as particles/cells pass through each opticalinterrogation zone.

FIG. 6 and FIG. 7 illustrate additional examples of the method of thepresent invention for detecting the modulated light emitted from threedifferent OIZs along a flow channel and for de-modulating and processingtotal detected electronic signals to recover and isolate theparticle-induced electronic pulse profiles as particles/cells passthrough each optical interrogation zone.

FIGS. 6A-F show the process of the analog modulation and de-modulationfor an example of three light sources, two of which areintensity-modulated and one of which is not modulated. FIGS. 6A and 6Bshow the analog signals used to modulate the 1^(st) and 3^(rd) lightsource, respectively. In this example, modulation frequency for the1^(st) and 3^(rd) light source is 3 MHz and 6 MHz, respectively. FIG. 6Cshows the total detected electronic pulse signal if no modulation isemployed for any of three light sources. A particle/cell passes throughthe 1^(st) OIZ, the 2^(nd) OIZ and the 3^(rd) OIZ sequentially andgenerate the 1^(st), 2^(nd) and 3^(rd) pulse, respectively. In thisexample, we assume that particle/cell diameter is 10 microns, passingthrough the OIZs with a 15 micron along the particle/cell flowingdirection, and the linear velocity of the particle/cell is assumed to be5 m/sec. Furthermore, we assume that the center-to-center distancebetween two adjacent OIZs is 30 microns. Therefore, the width of thegenerated individual particle-induced electronic pulse is estimated tobe 5 μs (i.e. (10 μm+15 μm)/(5 m/s)) and the separation between twopulse peaks is estimated to be 6 μs (i.e. 30 μm/(5 m/s). FIG. 6D showsthe total detected electronic signals when the 1^(st) and the 3^(rd)light sources are modulated using the modulation signals of FIG. 6A andFIG. 6B, respectively. Note that the 2^(nd) light source is notmodulated. FIGS. 6E and 6F illustrates the intermediate signal whende-modulation is applied to the total signal of FIG. 6D to recover the1^(st) pulse profile and 3^(rd) pulse profile, respectively. Theintermediate signals shown in FIG. 6E and FIG. 6F are calculated bymultiplying the detected electronic signals in FIG. 6D with themodulation sine-wave signal in FIG. 6A and FIG. 6B, respectively. FIGS.6G, 6H and 6I show the recovered the 1^(st), the 2^(nd) and the 3^(rd)electronic pulse profiles, respectively, generated as the particlepasses through the 1^(st), 2^(nd) and 3^(rd) light beams at three OIZs.The recovered electronic pulse signals are determined by low-passfiltering the intermediate de-modulation signals in FIG. 6E and FIG. 6Fand by low-pass filtering the total detected electronic signals in FIG.6C. Note that an amplification/scaling factor of 2 is applied to thesignals on FIGS. 6G and 6I after the signals at FIGS. 6E and 6F arefiltered by a low-pass filter with a cut-off frequency of 1 MHz.

FIG. 7 shows an additional example for the process of the analogmodulation and de-modulation for a case of three light sources, two ofwhich are intensity-modulated and one of which is not modulated.Different from the example in FIG. 6 where two light sources aremodulated at different modulation frequencies. The two light sourcesbeing modulated in FIG. 7 are modulated at the same modulationfrequency. FIG. 7A shows an analog sine-wave signal with a frequency of3 MHz used to modulate the 1^(st) and 3rd light source. FIG. 7B showsthe total detected electronic pulse signal if no modulation is employedfor any of three light sources. A particle/cell passes through the1^(st) OIZ, the 2^(nd) OIZ and the 3^(rd) OIZ sequentially and generatethe 1^(st), 2^(nd) and 3^(rd) pulse, respectively. In this example, weassume that particle/cell diameter is 10 microns, passing through theOIZs with a 15 micron along the particle/cell flowing direction, and thelinear velocity of the particle/cell is assumed to be 5 m/sec.Furthermore, we assume that the center-to-center distance between twoadjacent OIZs is 30 microns. Therefore, the width of the generatedindividual particle-induced electronic pulse is estimated to be 5 μs(i.e. (10 μm+15 μm)/(5 m/s)) and the separation between two pulse peaksis estimated to be 6 μs (i.e. 30 μm/(5 m/s). FIG. 7C shows the totaldetected electronic signals when the 1^(st) and the 3^(rd) light sourcesare modulated using the modulation signals of FIG. 7A. Note that the2^(nd) light source is not modulated. FIG. 7D illustrates theintermediate signal when de-modulation is applied to the total signal ofFIG. 7C to recover the 1^(st) and the 3^(rd) pulse profiles. Theintermediate signal shown in FIG. 7E is calculated by multiplying thedetected electronic signals in FIG. 7C with the modulation sine-wavesignal in FIG. 7A. FIG. 7E shows the recovered 1^(st) and 3^(rd)electronic pulse profiles, generated by low-pass filtering theintermediate de-modulation signals in FIG. 7D. FIGS. 7F, 7F and 7H showthe recovered 1^(st), 2^(nd) and 3^(rd) electronic pulse profiles,respectively, generated as the particle passes through the 1^(st),2^(nd) and 3^(rd) light beams at three OIZs. Note that anamplification/scaling factor of 2 is applied to the signals on FIGS. 7E,7F and 7H after the signals at FIG. 7D are filtered by a low-pass filterwith a cut-off frequency of 1 MHz. In this example, the same modulationsignal is used for modulating the 1^(st) and 3rd light sources. Becausethe modulated light beams are focused/directed to different OIZs (1^(st)OIZ and 3^(rd) OIZ) along the flow direction of the flow cell, it ispossible to separate, isolate and identify the 1^(st) and 3^(rd) pulseprofiles caused by particle traveling through the 1^(st) OIZ and the3^(rd) OIZ, based on identifying appropriate time windows. Compared withthe situation where the light sources are modulated with differentmodulation signals in FIG. 4 and FIG. 6, example shown in FIG. 7 wherethe same modulation signal and the same modulation frequency are used tomodulate two different light sources, offers a special advantage suchthat a same signal processing circuit and software can be applied tode-modulate the electronic signals generated at the photodetectorsassociated with fluorescent molecules excited by two different lightsources. This is beneficial to reducing over system complexity involvedin signal processing. By using only one set of de-modulation andsignal-recovery hardware and software for processing and de-modulatingsignals from two different light sources, more system resources could beavailable for processing various signals in the system, potentiallyimproving signal to noise ratio for all fluorescence detections. Inaddition, since only one modulation frequency is used, choice ofsuitable modulation frequency would be more straightforward, comparedwith the case where two or more light sources are modulated at differentmodulation frequencies. This is especially true and important when themodulation signals have waveforms other than sine waves.

The flow cytometer system in the present invention can be coupled with aparticle/cell sorting mechanism wherein particles or cells can bedirected or sorted into different outlet chambers or containers afterbeing detected at the optical interrogation zones. Such sorting would bebased on the measurement and detection of fluorescent signals atdifferent optical interrogation zones. Particles or cells of desiredproperties could be sorted together for further analysis or otherusages. Various mechanisms of particle sorting, as being used in variouscommercial flow cytometers, can be applied to the particle sorting inthe present invention. For example, in one approach, the flow ofparticles is controlled so that there is a large separation betweenparticles relative to their diameter. A vibrating mechanism causes thestream of particles (or cells) to break into individual droplets. Theparticle concentration, the flow speed, droplet size and otherparameters are adjusted so that there is a low probability of more thanone cell per droplet. Immediately before the particle stream breaks intodroplets, the particle flow passes through optical interrogation zoneswhere the fluorescent properties or characteristics of each particle isdetermined and measured. An electrical charging mechanism is used tocharge the droplet as it breaks from the stream, depending on whetherparticle being measured has the desired characteristics. The chargeddroplets then fall through an electrostatic deflection sub-system thatdiverts droplets into containers based upon their charge and thus upontheir characteristics.

The system and method of the present invention for detecting emittedlight excited by multiple light sources from multiple opticalinterrogation zones in a flow channel has a number of benefits.

Firstly, the approach does not require complex optical components/setupfor collecting and separating light from multiple optical interrogationzones into optically/physically different positions in space forfeasible detection. This would greatly simplify the optical collectionoptics setup and reduce the strict requirement for high accuracy ofadjustment and alignment of the light collection lenses and positions ofthe subsequent filters and photodetectors. This is an advantage over theapproach described in FIG. 1 where light from different OIZs need to bephysically/optically separated.

Secondly, the approach uses simplified light-collection and detectionsetup (i.e. the emitted light with the same detection wavelength can bedetected by the same set of filter and photodetector even though theyare excited by different light sources). This is an advantage over theapproach described in FIG. 1 where different optical filter andphotodetector is needed for detecting emitted light from different lightsources even though the detection wavelength range is the same.Simplified light-collection optics and detection setup reduces thecomplexity of the system and thus the cost.

Thirdly, light emitted from different OIZs due to different excitationlight sources can be detected and determined by exploiting modulationand de-modulation in electronics field, allowing the system to work withdifferent fluorescent molecules with same/overlapped emission spectra inthe same experiment. This is an advantage over the approach described inFIG. 2 where the system could not distinguish different fluorescentmolecules having same/overlapped emission spectra but excited bydifferent excitation light sources.

Fourthly, the modulation of the excitation light sources andde-modulation of the detected electronic signals is independent betweendifferent light sources. This is an advantage over the approachdescribed in U.S. Pat. No. 7,990,525 where accurate control of thetime-multiplexed illumination of multiple excitation light beams isessential and so as the subsequent synchronization of the signalprocessing electronics.

Furthermore, the system and method of the present invention fordetecting emitted light excited by multiple light sources from multipleoptical interrogation zones in a flow channel surprisingly achieves anumber of additional benefits.

Firstly, directing light beams from multiple excitation sources intodifferent OIZs at different locations along the flow channel in thepresent invention provides a further benefit that the full dynamic rangeof the photodetector and associated electronics circuits could beefficiently exploited/utilized for each analyzed fluorescent molecules.This is an advantage over the approach described in FIG. 2 wheremultiple excitation light beams are directed to the same location in theflow channel. In FIG. 2, unless at any given time moment, only one lightsource is activated (i.e. turned on), light emitted from different typesof fluorescent molecules having the same/overlapped emission spectra butexcited by different light sources would be mixed together and thecorresponding electronic pulses would be superimposed on each other.This leads to actual reduction of the upper limit of the detecting rangefor each of analyzed fluorescent molecules. For example, in FIG. 2,assume that two types of fluorescent molecules are used and they canonly be excited by two different light sources but emit fluorescentlight with overlapped spectra. If the condition (e.g., the number offluorescent molecules per particle/cell, the energy of light beams fromtwo excitation light sources, the fluorescence-generating efficiency ofthe fluorescent molecules, etc.) for these two types of fluorescentmolecules is similar, then the total detected signal from the samephotodetector would be the signal from both fluorescent molecules addedtogether. Since the dynamic range of the photodetector is a fixed value,the upper limit of the detecting range for each fluorescent signal wouldbe reduced by half assuming full dynamic range of the photodetector isused. For the present invention, fluorescent molecules with overlappedemission spectra will be excited at different OIZs at differentlocations along the flow channel. Thus, by properly choosing thecenter-to-center distance of adjacent light beams at different OIZs,probability of two particles/cells being both within two OIZs isminimized or very low. Therefore, at any given time moment, thephoto-detector only detects the emission light from one type offluorescent molecule, allowing for the efficient use of the full dynamicrange of the photodetector.

Secondly, directing light beams from multiple excitation light sourcesinto different OIZs at different locations along the flow channel in thepresent invention has a further benefit of improved sensitivity indetecting low-light-emitting, dimly-stained fluorescent particles/cells.This is an advantage over the approach described in FIG. 2 wheremultiple excitation light beams are directed to a single OIZ in the flowchannel. In FIG. 2, unless at any given time moment, only one lightsource is activated (i.e. turned on), light emitted from different typesof fluorescent molecules having the same/overlapped emission spectra butexcited by different light sources would be mixed together and thecorresponding electronic pulses would be superimposed on each other.Light emitted from one type of fluorescent molecule would become thebackground for light emitted from the other type(s) of fluorescentmolecules, and vice versa. Such “mutual background” of light emittedfrom different types of fluorescent molecules would reduce thesensitivity for detecting dimly stained fluorescent particles/cellsbecause it might be embedded in a strong fluorescent signal from anotherfluorescent molecule excited by another light source. For the presentinvention, light emitted from each type of fluorescent molecule is onlyexcited at a single OIZ at a single location of the flow channel. Thus,by properly choosing the center-to-center distance of adjacent lightbeams at different OIZs, probability of two particles/cells being bothwithin two OIZs is minimized or very low. The only background lightcomes from the stray light of the flow channel and the optical set up.Thus, detection of low or dim fluorescent signals could be possibleusing present invention.

Thirdly, the approach of different modulations applied to differentlight sources and the approach of recovering emitted light componentsdue to different light sources based on their modulation methods in thepresent invention has a further benefit that there is no veryaccurate/precise requirement for separation distance between differentOIZs. The de-modulation and electronic signal recovery means caneffectively recover the light-intensity-dependent electronic signalpulse due to each light source by effective modulation method for thecorresponding light source and as long as the light beams directed atdifferent OIZs are kept relatively stable over the time scale requiredfor a particle/cell passing through an OIZ. The small variation inseparation distance between different OIZs does not affect themeasurement results since the particle-induced electronic pulse signalscould be recovered through de-modulation and the positioning of suchpulses could be readily identified through an analysis algorithm. Thissimplifies the requirement for the optical subs-system fordelivering/directing light beams into different OIZs. This is anadvantage over the approaches shown in FIG. 1 and FIG. 2. In FIG. 1, itis essential to keep and maintain the different light beams at differentOIZs at an accurately controlled distance, since the variation in thisdistance would affect the efficiency of the light collection from eachOIZ, leading to unreliable measurement results of the fluorescentsignals. In FIG. 2, the different light beams from different excitationlight sources are required to be aligned coaxially to the same OIZ at asingle location in the flow channel. A complex optical setup is requiredto achieve such accurate control of the relative positions of the lightbeams. Any disturbance of the optical system, such as temperature,pressure, misalignment during instrument shipping, or other systeminstability factors would cause the beams not coaxially aligned andmight leads to misinterpretation of collected signals. On the otherhand, for the present application, there is no very accurate/preciserequirement for separation distance between different light beams (i.e.the separation distance between adjacent OIZs). Thus, the opticalsub-system for delivering light beams to the flow channel could besimplified and easy to maintain.

Fourthly, the present invention has a further benefit of achievinghigher signal-to-noise ratio due to the modulation/de-modulation methodit employed. As described above in several examples shown in Eqn (1)through Eqn (11), the de-modulation for recovering the particle-inducedelectronic pulse profile involves a low-pass filtering procedure basedon the bandwidth of the particle-induced electronic pulse. Such low-passfiltering can effectively remove the high-frequency noises signals inthe system, such as the thermal noises of the electronic elements.Therefore, the overall signal-to-noise ratio could be improved usingpresent invention.

What is claimed is:
 1. A system for detecting signal components of lightinduced by multiple excitation sources, the system comprising: a flowchannel configured for the flow of particles, the flow channelcomprising a first optical interrogation zone and a second opticalinterrogation zone, wherein the first optical interrogation zone and thesecond optical interrogation zone are spatially separated; a lightillumination subsystem configured to direct a first light beam to thefirst optical interrogation zone and to direct a second beam to thesecond optical interrogation zone, wherein the wavelengths of the firstlight beam are different from the wavelengths of the second light beam,wherein the first light beam is intensity modulated at a modulatingfrequency that is a constant pattern repeating over time and the secondlight beam is not intensity modulated to provide a constant intensity;shared light collection optics configured to collect light from both ofthe optical interrogation zones, wherein the light comprises a firstportion of light from the first interrogation zone and a second portionof light from the second interrogation zone, the first portion of lightbeing intensity modulated at the modulating frequency and resulting fromthe first beam being directed to the first optical interrogation zone;shared light splitting optics configured to split the collected lightinto different channels, wherein the shared light splitting opticscomprise a filter shared by each of the optical interrogation zones; adetector subsystem comprising a set of at least two light detectors andconfigured to receive the channeled light, detect changes in thechanneled light in response to particles flowing through the opticalinterrogation zones, and to convert the detected light into a totalelectrical signal proportional to detected modulated and unmodulatedlight; and a processor configured to receive the total electrical signalfrom the detector subsystem, to de-modulate the portion of the totalelectrical signal proportional to the modulated light, and to determinesignal components from the light detected from each of the opticalinterrogation zones.
 2. The system according to claim 1, wherein theoptical interrogation zones are spaced from about 30 to about 80 micronsapart.
 3. The system according to claim 1, wherein the lightillumination subsystem comprises a laser or a light emitting diode(LED).
 4. The system according to claim 1, wherein the wavelengths areselected from the group consisting of about 325 nm, 355 nm, 365 nm, 375nm, 405 nm, 407 nm, 488 nm, 532 nm, 561 nm, 595 nm, 633 nm, 635 nm, 640nm, and 647 nm.
 5. The system according to claim 1, wherein theintensity modulation is at a frequency between 1 MHz and 100 MHz.
 6. Thesystem according to claim 1, wherein the pattern is selected from thegroup consisting of a sine waveform, a square waveform, a triangularwaveform and a seesaw waveform.
 7. The system according to claim 1,wherein the set of detectors selectively detect wavelengths of about421±30 nm, 450±30 nm, 455±40 nm, 519±30 nm, 530±15 nm, 578±15 nm, 585±40nm, 603±30 nm, 615±30 nm, 620±30 nm, >650 nm, 660±10 nm, 667±30 nm,668±30 nm, 678±30 nm, 695±25 nm, >750 nm, 780±30 nm and >785 nm.
 8. Thesystem according to claim 1, further comprising a third spatiallyseparated optical interrogation zone and a third light beam directed tothe third optical interrogation zone.
 9. The system according to claim8, wherein the third light beam is intensity modulated over time at saidmodulation frequency.
 10. The system according to claim 1, furthercomprising a sorting mechanism configured to sort a flowing particlepopulation into one or more chambers in response to commands from theprocessor.
 11. The system according to claim 1, wherein the modulatedintensity is modulated according to a waveform selected from the groupconsisting of a sine waveform, a square waveform, a triangular waveformand a seesaw waveform.
 12. A method of detecting signal components fromlight induced by multiple excitation sources, the method comprising:providing a flow channel comprising a first optical interrogation zoneand a second optical interrogation zone, wherein the first and secondoptical interrogation zones are spatially separated; flowing apopulation of particles labeled with at least two different fluorescentmolecules through each of the first and second optical interrogationzones, wherein the at least two different fluorescent molecules comprisea first fluorescent molecule and a second fluorescent molecule;directing at a constant intensity and to the first optical interrogationzones, a first light beam comprising a wavelength that inducesfluorescence of the first fluorescent molecules; directing at amodulated intensity and to the second optical interrogation zones, asecond light beam comprising a wavelength that induces fluorescence ofthe first or second fluorescent molecule, wherein the modulatedintensity is a constant pattern repeating over time; collecting lightfrom both of the optical interrogation zones with shared collectionoptics, wherein the light comprises a first portion of light from thefirst interrogation zone and a second portion of light from the secondinterrogation zone, the intensity of the second portion of light beingmodulated at the constant pattern and resulting from the second beambeing directed to the second optical interrogation zone; splitting thecollected light into different channels using a filter shared by thelight collected from each of the optical interrogation zones; detectingthe split light; converting the detected light into a total electricalsignal proportional to detected modulated and unmodulated light;de-modulating the portion of the total electrical signal proportional tothe modulated light; and determining signal components of the lightdetected from each of the optical interrogation zones.
 13. The methodaccording to claim 12, wherein the modulated intensity is at a frequencybetween 1 MHz and 100 MHz.
 14. The method according to claim 12, whereinthe fluorescent molecules emit a wavelength selected from one or more ofthe group consisting of 519 nm, 578 nm, 640 nm, 660 nm, and 785 nm. 15.The method according to claim 12, wherein the flow channel furthercomprises a third optical interrogation zone, and the method furthercomprises directing a third light beam to the third opticalinterrogation zone, wherein the third light beam is intensity modulatedand comprises a wavelength that induces fluorescence of a thirdfluorescent molecule.
 16. The method according to claim 12, furthercomprising sorting the cell population into one or more chambersaccording to the presence or absence of one or more detected components.